Fungi adapted to metabolize phosphite as a source of phosphorus

ABSTRACT

System, including methods and compositions, for making and using fungi that are adapted transgenically to metabolize phosphite as a source of phosphorus for supporting growth.

CROSS-REFERENCES TO PRIORITY APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/130,285, filed May 19, 2011, which in turn claims priority under 35U.S.C. §371 to PCT Application Serial No. PCT/IB2009/007741, filed Nov.19, 2009, which in turn claims priority to U.S. Provisional PatentApplication Ser. No. 61/199,784, filed Nov. 19, 2008.

Each of these priority documents is incorporated herein by reference inits entirety for all purposes.

INTRODUCTION

Phosphorus is an essential element for plant and fungal growth. Thiselement, in oxidized form, is incorporated into many of the biomoleculesin a plant or fungal cell, such as to provide genetic material,membranes, and molecular messengers, among others.

Inorganic phosphate (Pi) is the primary source of phosphorus for plants.Accordingly, phosphate-based fertilizers offer a cheap and widely usedapproach to enhancing plant growth. However, phosphate-based fertilizerscome from a non-renewable resource that has been projected to bedepleted in the next seventy to one hundred years, or sooner if theusage rate increases faster than expected.

The phosphate-based fertilizers common to modern agriculture generallycannot be used efficiently by cultivated plants, due to severalimportant factors. First, phosphate is highly reactive and can forminsoluble complexes with many soil components, which reduces the amountof available phosphorus. Second, soil microorganisms can rapidly convertphosphate into organic molecules that generally cannot be metabolizedefficiently by plants, which reduces the amount of available phosphorusfurther. Third, growth of weeds can be stimulated by phosphate-basedfertilizers, which not only reduces the amount of available phosphorusstill further but which also can encourage the weeds to compete with thecultivated plants for space and other nutrients. Losses due to theconversion of phosphate into inorganic and organic forms that are notreadily available for plant uptake and utilization, and competition fromweeds, implies the use of excessive amounts of phosphate fertilizer,which not only increases production costs but also causes severeecological problems. Therefore, there is an urgent need to reduce theamount of phosphate fertilizer used in agriculture.

A reduced form of phosphate, phosphite (Phi), is also used incultivation of plants. It has been shown that treatment with phosphitecan increase plant production (as measured by fruit size and biomass) inavocado and citrus fruits. Phosphite may be transported into plantsusing the same transport system as phosphate and may accumulate in planttissues for extended periods of time. However, there apparently are noreports of any enzymes in plants that can metabolize phosphite intophosphate, the primary source of phosphorus in plants. Moreover, evenduring phosphate starvation, phosphite apparently cannot satisfy thephosphorus nutritional requirements of the plant. Accordingly, in spiteof similarities to phosphate, phosphite is a form of phosphorus thatgenerally cannot be metabolized directly by plants, and thus is not aplant nutrient. Nevertheless, phosphite “fertilizers” are soldcommercially, even though there appears be no proof or even anindication in the scientific literature that plants can assimilatephosphite.

Phosphite can promote plant growth indirectly. For example, phosphite isused as an anti-fungal agent (a fungicide) on cultivated plants.Phosphite is thought to prevent diseases caused by oomycetes (watermolds) on such diverse plants as potato, tobacco, avocado, and papaya,among others. Phosphite thus may promote plant growth, not directly as aplant nutrient, but by protecting plants from fungal pathogens thatwould otherwise affect plant growth. Nevertheless, phosphite-basedfungicides often are labeled as fertilizers. This mislabeling may beencouraged by government regulations that make the approval processshorter and less complex if manufacturers characterize fungicides asfertilizers.

The proposed mechanisms for phosphite acting as a fungicide aremanifold. For example, phosphite may act on fungi by inhibitingphosphorylation reactions through an increment in the accumulation ofinorganic pyrophosphate (PPi), which in turn can interrupt phosphatepathways that are metabolically critical. Alternatively, or in addition,phosphite may induce a natural defense response in plants. In any event,the efficacy of phosphite as a fungicide may be influenced by severalfactors, including environment, type of pathogen, type of plant, andconcentration.

The concentration of phosphite in contact with plants may be a criticalfactor for phosphite effectiveness because too much phosphite can betoxic to plants. In particular, phosphite may compete with phosphate forentry into plant cells, since phosphite may be transported into plantsvia the phosphate transport system. Phosphite toxicity thus may be dueto (1) reduced assimilation of phosphate by plants, in combination with(2) an inability to use phosphite as a source of phosphorus by oxidationto phosphate, which causes phosphite accumulation in the plants. Also,phosphite may be sensed in plants as phosphate, which prevents theplants from inducing a phosphorus salvage pathway that promotes plantsurvival under conditions of low phosphate. Phosphite toxicity affectssuch diverse plants as Brassica nigra, Allium cepa (onion), Zea mays L.(corn), and Arabidopsis thaliana. Accordingly, the exposure of plants tophosphite may need to be controlled very carefully. Therefore, a bettersystem is needed for exploiting the benefits of phosphite to plantswhile reducing its drawbacks.

Generation of transgenic plants has been instrumental in creatingimproved agricultural systems. At least four selection systems have beenestablished for identifying transgenic plants by selective growth. Eachselection system is based on resistance to an antibiotic (kanamycin orhygromycin) or an herbicide (glyphosate or phosphinothricin). However,each selection system has disadvantages. For example, each selectionsystem can have problems with toxicity. Also, selection with antibioticsmay be inefficient since plants can have alternate resistancemechanisms. Furthermore, except for the selection system usingphosphinothricin, none of the selection systems provides a “universal”selectable marker for most or all plants. Therefore, a new selectablemarker is needed for use in generating transgenic plants.

SUMMARY

The present disclosure provides a system, including methods andcompositions, for making and using fungi that are adapted transgenicallyto metabolize phosphite as a source of phosphorus for supporting growth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic flowchart of an exemplary method of (i) making atransgenic plant (or fungus) that is capable of metabolizing a reducedform of phosphorus, such as phosphite, as a source of phosphorus forsupporting growth, and/or (ii) using, as a selectable marker, a nucleicacid that confers a capability to metabolize a reduced form ofphosphorus, such as phosphite, as a source of phosphorus for supportinggrowth, in accordance with aspects of the present disclosure.

FIG. 2 is a schematic representation an exemplary nucleic acid for usein the method of FIG. 1, in accordance with aspects of the presentdisclosure.

FIG. 3 is a proposed mechanism for oxidation of hypophosphite tophosphate in bacteria using enzymes expressed from the ptxD and htxAgenes of Pseudomonas stutzeri, in accordance with aspects of the presentdisclosure.

FIG. 4 is a schematic representation of an exemplary chimeric geneconstructed for use in generating a transgenic plant that metabolizesphosphite to phosphate, in accordance with aspects of the presentdisclosure.

FIG. 5 is a schematic diagram of a portion of a strategy followed tocreate the chimeric gene of FIG. 4, in accordance with aspects of thepresent disclosure.

FIG. 6 is a pair of photographs showing exemplary data obtained with thechimeric gene of FIG. 4 used as a selectable marker by selection oftransgenic plants for their ability to grow on a phosphite-containingmedium in the absence of phosphate, in accordance with aspects of thepresent disclosure.

FIG. 7 is a series of photographs of data obtained from growth tests ofcontrol and transgenic (ptxD) Arabidopsis lines germinated andcultivated in a liquid growth medium, with or without phosphite (Phi) orphosphate (Pi) as the source of phosphorus, in accordance with aspectsof the present disclosure.

FIG. 8 is a bar graph of data obtained from tests of the ability of theArabidopsis lines of FIG. 7 to extract phosphorus from their growthmedia, with the plants cultivated for 45 days in growth media containingdifferent concentrations of phosphite (Phi) as the source of phosphorus,in accordance with aspects of the present disclosure.

FIG. 9 is a schematic representation of the distribution of control (WT)and ptxD transgenic (PTXD) Arabidopsis plants across a growth substrate,as used for the experiments of FIGS. 10 and 11, in accordance withaspects of the present disclosure.

FIG. 10 is a photograph of parental and ptxD transgenic plantsdistributed according to FIG. 9 and tested for growth on a substratecontaining phosphate (Pi) as the source of phosphorus, in accordancewith aspects of the present disclosure.

FIG. 11 is a photograph of parental and ptxD transgenic plantsdistributed according to FIG. 9 and tested for growth on a substratecontaining phosphite (Phi) as the source of phosphorus, in accordancewith aspects of the present disclosure.

FIG. 12 is a bar graph of data obtained from tests of the ability of theArabidopsis lines of FIG. 7 to increase in weight when cultivated in theabsence or presence of phosphate and/or phosphite as the source ofphosphorus, in accordance with aspects of the present disclosure.

FIG. 13 is a set of photographs of control (WT) and ptxD transgeniclines of Nicotiana tabacum (tobacco) 25 days after germination in thepresence of phosphate or phosphite as the source of phosphorus, inaccordance with aspects of the present disclosure.

FIG. 14 is a set of photographs of another growth experiment performedwith the control and transgenic tobacco lines of FIG. 13, in accordancewith aspects of the present disclosure.

DETAILED DESCRIPTION

The present disclosure provides a system, including methods andcompositions, for making and using transgenic plants and/or transgenicfungi that metabolize phosphite as a source of phosphorus for supportinggrowth. The plants and/or fungi optionally also may metabolizehypophosphite as a source of phosphorus. The system disclosed herein maysubstantially change the way a more reduced form of phosphorus (relativeto phosphate), such as phosphite, is utilized as a fertilizer and/orfungicide. The system also may provide a new selectable marker for usein generating transgenic plants and/or fungi. The system further maysubstantially change the way at least one reduced form of phosphorus isremoved from waste water, such as industrial/municipal effluents.

A nucleic acid is provided. The nucleic acid may be used for generatinga transgenic plant and/or fungus. The nucleic acid, which may be termeda construct, may comprise at least one chimeric gene that confers on aplant cell and/or fungal cell a capability to metabolize at least onereduced form of phosphorus to phosphate. In some embodiments, thenucleic acid may comprise a gene that expresses a phosphitedehydrogenase enzyme, a gene that expresses a hypophosphitedehydrogenase enzyme, or both.

The nucleic acid may comprise a chimeric gene including a coding regionand a transcription promoter. The coding region may encode a phosphitedehydrogenase enzyme, such as PtxD from Pseudomonas stutzeri, a homologof PtxD from the same or another bacterial species, or a derivative ofeither, among others. In some examples, the coding region may be atleast 80%, 90%, or 95% (or completely) identical to the ptxD codingsequence of Pseudomonas stutzeri. The promoter may be functional inplants, fungi, or both and may be operatively linked to the codingregion. The promoter may be heterologous with respect to the codingregion. The chimeric gene may be capable of promoting sufficientexpression of the enzyme, in a plant or fungal cell containing thenucleic acid, to confer an ability on the cell to metabolize phosphite(Phi) as a phosphorus source for supporting growth, thereby enablinggrowth of the cell without an external source of phosphate (Pi). Thepromoter may (or may not) be a plant promoter or a viral promoter of aplant virus and may be capable of promoting the sufficient expression ofthe enzyme in a plant cell. For example, the promoter, such as apromoter obtained from the PLDZ2 gene of Arabidopsis thaliana, may beinducible by low phosphate availability. Alternatively, or in addition,the promoter may be a root-specific promoter. In other cases, thepromoter may be constitutive and may correspond to the 35S promoter ofCauliflower Mosaic Virus. In some embodiments, the nucleic acid mayinclude a transcription terminator that is functional in the plant celland/or fungal cell and that is operatively linked to the promoter andcoding region. In some embodiments, the promoter may be a fungalpromoter capable of promoting the sufficient expression of the enzyme ina fungal cell.

The nucleic acid may provide expression of one or more polypeptides thatmetabolize at least one reduced form of phosphorus to phosphate, toenable a transgenic plant (or fungus) to use a reduced form ofphosphorus as a nutrient. The expression of the one or more polypeptidesmay be heritable. For example, the nucleic acid may be integrated intothe genome of the plant (or fungus). Furthermore, the expression of atleast one of the polypeptides may be under control of a constitutivepromoter or an inducible promoter (e.g., inducible by low phosphate,such as by use of a promoter from a PLDZ2 gene of Arabidopsis or a plantAtPT1 gene for a high affinity phosphate transporter), under control ofa tissue-specific promoter (e.g., leaf-specific or root-specific), or acombination thereof, among others.

A plant cell or a fungal cell is provided that expresses a phosphitedehydrogenase enzyme from a chimeric gene. The cell may be isolated fromother cells or may be associated with other cells in a multi-cellularstructure (e.g., a plant or a mycelium). The cell may (or may not) alsoexpress a hypophosphite dehydrogenase enzyme from a chimeric gene.Accordingly, the cell may metabolize phosphite, hypophosphite, or both,as a phosphorus source for supporting growth. In some embodiments, thecell may be a plant cell and expression of the phosphite dehydrogenaseenzyme, the hypophosphite dehydrogenase enzyme (if present), or both maybe controlled by a root-specific promoter. The plant cell may be fromany suitable species. For example, the plant cell may be a eukaryoticalgal cell, such as a Chlamydomonas cell. In other cases, the plant cellmay be from a species of vascular plant. In some embodiments, the cellmay be a fungal cell that belongs to a species of Trichoderma or thatbelongs to a mycorrhizal species of fungus capable of forming asymbiotic relationship with a plant.

A transgenic plant (or plant part) is provided that expresses aphosphite dehydrogenase enzyme, and, optionally, a hypophosphitedehydrogenase enzyme from one or more chimeric genes. The plant may,through expression of the enzyme(s), metabolize phosphite and/orhypophosphite as a source of phosphorus for supporting growth. The plantmay be a vascular plant, such as crop plant, for example, a species ofcrop plant selected from the group consisting of maize, soybean, rice,potatoes, tomatoes, sugarcane, and wheat. A seed that germinates toproduce the transgenic plant also is provided.

A method of reducing fungal infections in plants is provided. Aplurality of fungal cells may be applied to a seed form of plants, theplants themselves, soil in which the plants are or will be disposed, ora combination thereof. The fungal cells may express a phosphitedehydrogenase enzyme from a chimeric gene and may belong to a species ofTrichoderma.

A plant associated with a plurality of fungal cells to form mycorrhizaeis provided. The fungal cells may express a phosphite dehydrogenaseenzyme from a chimeric gene. The fungal cells may render the plantcapable of growth on phosphite (and/or hypophosphite) as a phosphorussource by oxidizing phosphite to phosphate.

A method is provided of fertilizing a crop plant using hypophosphiteand/or phosphite as a phosphorus source for supporting growth. The cropplant may express a phosphite dehydrogenase enzyme, a hypophosphitedehydrogenase enzyme, or both. Alternatively, or in addition, the cropplant may form mycorrhizae with a plurality of fungal cells expressing aphosphite dehydrogenase enzyme, a hypophosphite dehydrogenase enzyme, orboth. At least one reduced form of phosphorus, such as phosphite and/orhypophosphite, may be applied to the plant and/or to soil adjacent theplant, such that the reduced form is metabolized to phosphate by theplant and/or the mycorrhizae to support growth and productivity of theplant.

A method is provided of treating liquid waste (e.g., an effluent) tolower its content of reduced phosphorus. Contact is created between (i)water containing hypophosphite and/or phosphite as a contaminant and(ii) a plurality of plant cells and/or fungal cells expressing aphosphite dehydrogenase enzyme, a hypophosphite dehydrogenase enzyme, orboth, such that at least a portion of the hypophosphite and/or phosphiteis oxidized to phosphite and/or phosphate. In some cases, the contactmay be created between the water and a plurality of vascular plantsexpressing one or both of the enzymes. The method may provide abioremediation system for rivers, reservoirs, soils, holding tanks, andthe like that are contaminated due to industrial manufacturing. Forexample, phosphite is a common polluting agent in rivers and lakes nearindustrial sites, such as manufacturers of optical discs (e.g., DVDs andCDs) that use hypophosphite to reduce metal ions in chemical platingprocesses. Transgenic plants and/or fungi disclosed herein thus may helpremove hypophosphite and/or phosphite from contaminated water by takingup and converting the hypophosphite and/or phosphite into phosphate. Useof plants and/or fungi may be more efficient than using abacterial-based system.

A method is provided of utilizing a coding sequence for a phosphitedehydrogenase as a selectable marker for production of a transgenicplant. The method may be used to obtain a plant transformed with anucleic acid encoding a phosphite dehydrogenase enzyme that isexpressible from the nucleic acid as a selectable marker. Plant cellsand a composition including the nucleic acid may be contacted underconditions that promote introduction of the nucleic acid into the plantcells. The plant cells may be cultured in a medium containing phosphiteas a primary or exclusive phosphorus source for growth of the plantcells. Selection may be performed of transformed plant cells produced bythe steps of contacting and culturing and that express the phosphitedehydrogenase enzyme as evidenced by growth in the medium. At least aportion of the transformed plant cells may be regenerated into atransgenic plant.

The transgenic plants disclosed herein may provide substantial benefits.For example, in some cases, the plants may metabolize phosphite usingNAD+ as an electron acceptor, to generate NADH and phosphate, which areboth useful molecules for the plant. The transgenic plants also oralternatively may provide development of a new agricultural system basedon phosphite. Phosphite may be less reactive in the soil than phosphateand therefore may create fewer insoluble compounds that the plant cannotutilize. Also, since most soil microorganisms are unable to metabolizephosphite, less of the phosphite (relative to phosphate) is convertedinto organic forms that plants cannot utilize. Furthermore, phosphitemay have less impact on the bacterial ecosystem around the plantsrelative to phosphate. Competition from weeds also may be reducedsubstantially since the weeds should not be able to utilize phosphite.The use of phosphite thus should decrease fertilizer costs and reducethe negative impact of fertilizer on the environment.

The transgenic plants disclosed herein also may offer increasedeffectiveness of phosphite as a fungicide, while acting as a fertilizeron the transgenic plants. When used as a fungicide on non-transgenicplants, phosphite generally needs to be used very carefully, to avoidplant toxicity. However, in the transgenic plants disclosed herein,phosphite may be metabolized by the plant to become non-toxic.

The system disclosed herein may provide substantial advantages forgenerating transgenic plants. A selectable marker of the system mayfunction at least substantially universally in plants. Furthermore, theselective agent (e.g., hypophosphite or phosphite) may be nontoxic fortransgenic plants, since the reaction products may be innocuous (e.g.,NADH and phosphate), and also may be less expensive than in otherselection schemes.

Further aspects of the present disclosure are provided in the followingsections: (I) definitions, (II) generation of transgenic plants andfungi, (III) use of transgenic plants and fungi, and (IV) examples.

I. DEFINITIONS

The various terms used in the present disclosure generally each have ameaning recognized by those skilled in the art, consistent with thecontext in which each term is used. However, the following terms mayhave additional and/or alternative meanings, as described below.

Plant—a member of the Plantae kingdom of eukaryotic organisms, which maybe described as a tree, bush, grass, shrub, herb, vine, fern, moss, aeukaryotic alga, or a combination thereof, among others. A planttypically possesses cellulose cell walls and is capable of carrying outphotosynthesis. The plant may be a vascular plant. In some embodiments,the plant may be an annual or a perennial. The plant may be a floweringplant, such as a monocotyledon or a dicotyledon. In some embodiments,the plant may produce a grain, tuber, fruit, vegetable, nut, seed,fiber, or a combination thereof, among others. Furthermore, the plantmay be a crop plant, which may be cultivated in a field. Exemplary cropplants that may be suitable for generation of transgenic plantsaccording to the present disclosure include tobacco (e.g., N. tabacum),potato, maize, rice, wheat, alfalfa, soybean, tomato, sugarcane, and thelike.

Plant part—any portion of a plant that is less than a whole plant andthat includes at least one plant cell. A plant part thus may be a planttissue, such as leaf tissue, root tissue, stem tissue, shoot tissue,callus tissue, flower tissue, or any combination thereof, among others.A plant part may be an isolated plant cell or a colony or set of plantcells. A plant cell may be a protoplast or may include a cell wall,among others.

Transgenic—comprising a nucleic acid construct. The construct may beintegrated into an organism's (and/or cell's) genome (e.g., nuclear orplastid genome), in any subset or at least substantially all of thecells of the organism. For example, the construct may be present in aplant's germline. Accordingly, the construct may be heritable, that is,inherited by at least one or more members, or at least substantially allmembers, of a succeeding generation of the organism, or in descendantsof a cell. A plant or fungus (or plant or fungal part (e.g., a cell))that is “transformed” with a construct has been modified to contain theconstruct in the current generation or in any preceding generation(s) ofthe plant or fungus (or plant or fungal part). A transgenic plant may beprovided by a seed that germinates to form the transgenic plant. Also, atransgenic plant may produce one or more seeds that germinate to producetransgenic progeny plants.

Nucleic acid—a compound comprising a chain of nucleotides. The chain maybe composed of any suitable number of nucleotides, such as at leastabout 10, 100, or 1000, among others. A nucleic acid may be termed apolynucleotide, and may, for example, be single-stranded,double-stranded, or a combination thereof.

Gene—a nucleic acid or segment thereof that provides an expressible unitfor expression of a polypeptide and/or a functional RNA (e.g., amessenger RNA, an interfering RNA, or an enzymatic RNA, among others). Agene thus may include (a) a coding region (also termed a codingsequence, which may be continuous or interrupted (such as by one or moreintrons)) to define the sequence of the polypeptide and/or functionalRNA, (b) at least one transcription promoter (also termed a promotersequence) and, (c) optionally, at least one transcription terminator(also termed a termination sequence), with the transcription promoterand the transcription terminator operatively linked to the codingregion. A gene optionally may include one or more other control regionsand/or untranslated regions, such as at least one 5′ untranslatedregion, 3′ untranslated region, intron, or any combination thereof,among others.

Promoter—a nucleic acid region that controls (i.e., promotes, regulates,and/or drives) transcription of a gene to produce a primary transcriptand/or a messenger RNA. A promoter may operate, for example, bydetermining, at least in part, the rate of transcriptional initiation ofa gene by RNA polymerase. The promoter also or alternatively maydetermine the rate of transcriptional elongation after transcription isinitiated. The promoter may be functional in plants and/or fungi andthus may be a plant promoter and/or a fungal promoter.

Chimeric gene—a gene with sequence elements, such as a transcriptionpromoter and a coding region, that are heterologous with respect to oneanother. The term “heterologous” means that the sequence elements (e.g.,the promoter and coding region) originate and/or are derived fromrespective distinct sources, such as distinct species of organisms. Achimeric gene also may comprise a transcription terminator, which mayoriginate from a source distinct from the coding region, and from thesame source as, or a source distinct from, the promoter. Exemplaryterminators that may be used in the chimeric genes include the 35Sterminator of Cauliflower Mosaic Virus, the nopaline synthase terminatorof Agrobacterium tumefaciens, or the like.

Construct—a nucleic acid created, at least in part, using techniques ofgenetic engineering. A construct thus may be termed a nucleic acidconstruct.

Expression—a process by which a product, namely, an RNA and/or apolypeptide, is formed from information provided by a nucleic acidand/or gene, generally in the form of DNA. Accordingly, the nucleicacid/gene may be expressed to form an RNA and/or polypeptide, whichmeans that the RNA and/or polypeptide is expressed from the nucleicacid/gene.

Reduced forms of phosphorus—any phosphorus-containing compounds and/orions in which phosphorus has an oxidation state of less than +5, such as+3 or +1. Accordingly, reduced forms of phosphorus may, for example,include phosphite and hypophosphite, among others. A reduced form ofphosphorus may be abbreviated “RP.”

Phosphate—phosphoric acid (H₃PO₄), its dibasic form (H₂PO₄ ¹⁻), itsmonobasic form (HPO₄ ²⁻), its triply ionized form (PO₄ ³⁻), or anycombination thereof. Phosphate may be provided as any suitable phosphatecompound or combination of phosphate compounds. Exemplary forms ofphosphate include phosphate salts of sodium, potassium, lithium,rubidium, cesium, ammonium, calcium, or magnesium, or any combinationthereof, among others. In phosphate, four oxygens are bonded directly toa phosphorus atom. Phosphate also or alternatively may be called“orthophosphate” and/or “inorganic phosphate” and may be abbreviated as“Pi.” Phosphate is distinct from “organophosphate,” which is an organicversion of phosphate in which one or more of the phosphate oxygens arebonded to organic moieties, generally to form a phosphate ester.

Phosphite—phosphorous acid (H₃PO₃), its conjugate base/singly ionizedform (H₂PO₃ ¹⁻), or its doubly ionized form (HPO₃ ²⁻), or anycombination thereof. In phosphite, three oxygens and one hydrogen arebonded directly to a phosphorus atom. Phosphite may be provided as anysuitable phosphite compound or combination of phosphite compounds.Exemplary forms of phosphite include phosphite salts of sodium,potassium, lithium, rubidium, cesium, ammonium, calcium, or magnesium,or any combination thereof, among others. Phosphite can be oxidized tophosphate. Phosphite also or alternatively may be called “inorganicphosphite” and may be abbreviated as “Phi.” Phosphite is distinct from“organophosphite,” which is an organic version of phosphite in which oneor more of the phosphite oxygens are bonded to organic moieties,generally to form a phosphite ester.

Hypophosphite—hypophosphorous acid (H₃PO₂) and/or its conjugate base(H₂PO₂ ⁻), which may be provided as any suitable hypophosphite compoundor combination of hypophosphite compounds. In hypophosphite, two oxygensand two hydrogens are bonded directly to a phosphorus atom. Exemplaryforms of hypophosphite include hypophosphite salts of sodium, potassium,lithium, rubidium, cesium, ammonium, or a combination thereof, amongothers. Hypophosphite can be oxidized to phosphite and/or to phosphate.Hypophosphite also or alternatively may be called “inorganichypophosphite” and may be abbreviated as “Hphi.”

Nutrient—any substance that is metabolized to promote growth andreproduction, and/or is required for survival.

Fertilizer—any composition that includes one or more nutrients forplants (and/or fungi associated with the plants).

External Source—a supply that is outside of a plant and accessible tothe plant, generally by contact with the plant. Exemplary externalsources that may be suitable for the transgenic plants described hereinmay include an external source of phosphorus, an external source ofphosphate, or an external source of reduced phosphorus, among others.

Selectable Marker—a construct or segment thereof and/or a gene thatconfers a growth advantage on a plant or plant part (and/or a fungusand/or fungal cell) that contains the construct/gene, when growth of theplant or plant part (and/or fungus and/or fungal cell) is tested bycontact with a suitable culture medium.

Effluent—water carrying and/or mixed with waste material. An effluentmay or may not be flowing. An exemplary effluent may, for example, beindustrial refuse and/or sewage, which may be combined with a largerbody of water, such as a stream, river, pond, lake, swamp, wetland, orthe like.

Remediation—any process that modifies water (e.g., waste water and/or aneffluent) to a more desired composition, such as to make the water lesstoxic, more environmental friendly, in better conformation withgovernment standards, etc.

Enzyme that oxidizes a reduced form of phosphorus—an enzyme thatcatalyzes or promotes oxidation of a reduced form of phosphorus (e.g.,with an oxidation state of +1 or +3) to a more oxidized state (e.g., +1to +3, +1 to +5, and/or +3 to +5). For example, the enzyme may oxidizehypophosphite to phosphite, phosphite to phosphate, and/or hypophosphiteto phosphate, among others. For convenience, the enzyme may be termed an“oxidase,” since it catalyzes/promotes an oxidation reaction, or may becalled a “phosphorus oxidoreductase” or “enzyme of reduced phosphorusmetabolism,” and may be abbreviated, for convenience herein, as“RP-OxRe.” Exemplary enzymes that oxidize a reduced form of phosphorusmay include a phosphite dehydrogenase enzyme (which may, for example, becalled NAD:phosphite oxidoreductase, phosphonate dehydrogenase,NAD-dependent phosphite dehydrogenase, or the like), a hypophosphitedehydrogenase (e.g., hypophosphite:2-oxoglutarate oxidoreductase), orthe like. The enzyme may oxidize a reduced form of phosphorus using anysuitable cofactor(s), coenzyme(s), and/or substrate(s) present in and/ornear a cell. Furthermore, the enzyme may originate and/or be derivedfrom bacteria, fungi, plants, or animals.

Phosphite dehydrogenase enzyme—an enzyme that catalyzes oxidation ofphosphite to phosphate. The enzyme generally catalyzes the oxidationwith sufficient efficiency to enable growth of a plant cell and/orfungal cell in the presence of phosphite as a phosphorus source tosupport growth. The enzyme may be of bacterial origin. The enzyme may bea PtxD polypeptide (i.e., PtxD or PtxD-like), which is any polypeptidethat is capable of catalyzing oxidation of phosphite to phosphate andthat is (a) at least 90%, 95%, or completely identical to PtxD (SEQ IDNO:1; GenBank: AAC71709.1) of Pseudomonas stutzeri WM 88, (b) aderivative of PtxD of SEQ ID NO:1, (c) a homolog (i.e., a paralog orortholog) of PtxD (SEQ ID NO:1) from the same or a different bacterialspecies, or (d) a derivative of (c). Homologs of PtxD (SEQ ID NO:1) havesubstantial similarity to PtxD of Pseudomonas stutzeri, which may, forexample, be determined by the blastp algorithm (e.g., program BLASTP2.2.18+), as described in the following two references, which areincorporated herein by reference: Stephen F. Altschul, et al. (1997),“Gapped BLAST and PSI-BLAST: a new generation of protein database searchprograms,” Constructs Res. 25:3389-3402; and Stephen F. Altschul et al.(2005) “Protein database searches using compositionally adjustedsubstitution matrices,” FEBS J. 272:5101-5109. Examples of substantialsimilarity include at least 50%, 60%, 70%, or 80% sequence identity, asimilarity score of at least 200 or 250, and/or an E-Value of less than1e-40, 1e-60, or 1e-80, among others, using the blastp algorithm, withoptimal alignment and, if needed, introduction of gaps.

Exemplary homologs of PtxD of Pseudomonas stutzeri may be provided byAcinetobacter radioresistens SK82 (SEQ ID NO:2; GenBank EET83888.1);Alcaligenes faecalis (SEQ ID NO:3; GenBank AAT12779.1); Cyanothece sp.CCY0110 (SEQ ID NO:4; GenBank EAZ89932.1); Gallionella ferruginea (SEQID NO:5; GenBank EES62080.1); Janthinobacterium sp. Marseille (SEQ IDNO:6; GenBank ABR91484.1); Klebsiella pneumoniae (SEQ ID NO.7; GenbankABR80271.1); Marinobacter algicola (SEQ ID NO:8; GenBank EDM49754.1);Methylobacterium extorquens (SEQ ID NO:9; NCBI YP_(—)003066079.1);Nostoc sp. PCC 7120 (SEQ ID NO:10; GenBank BAB77417.1); Oxalobacterformigenes (SEQ ID NO.11; NCBI ZP_(—)04579760.1); Streptomyces sviceus(SEQ ID NO:12; GenBank EDY59675.1); Thioalkalivibrio sp. HL-EbGR7 (SEQID NO:13; GenBank ACL72000.1); and Xanthobacter flavus (SEQ ID NO:14;GenBank ABG73582.1), among others. Further aspects of PtxD homologs aredescribed in U.S. Patent Application Publication No. 2004/0091985 (“the'985 publication”) to Metcalf et al., which is incorporated herein byreference. The phosphite dehydrogenase may have an amino acid sequencewith at least 50%, 60%, 80%, 90% or 95% or 100% sequence identity to oneor more of SEQ ID NOS:1-14.

Exemplary derivatives of PtxD of Pseudomonas stutzeri that may besuitable are described in the '985 publication and in U.S. Pat. No.7,402,419 to Zhao et al., which is incorporated herein by reference. Thederivatives may provide, for example, altered cofactoraffinity/specificity and/or altered thermostability.

The phosphite dehydrogenase enzyme may contain a sequence region withsequence similarity or identity to any one or any combination of thefollowing consensus motifs: an NAD-binding motif having a consensussequence of VGILGMGAIG (SEQ ID NO:15); a conserved signature sequencefor the D-isomer specific 2-hydroxyacid family with a consensus sequenceof XPGALLVNPCRGSVVD (SEQ ID NO:16), where X is K or R, or a shorterconsensus sequence within SEQ ID NO:16 of RGSVVD (SEQ ID NO:17); and/ora motif that may enable hydrogenases to use phosphite as a substrate,with a general consensus of GWQPQFYGTGL (SEQ ID NO:18), but that can bebetter defined as GWX₁PX₂X₃YX₄X₅GL (SEQ ID NO.19), where X₁ is R, Q, T,or K, X₂ is A, V, Q, R, K, H, or E, X₃ is L or F, X₄ is G, F, or S, andX₅ is T, R, M, L, A, or S. Further aspects of consensus sequences foundby comparison of PtxD and PtxD homologs are described in U.S. PatentApplication Publication No. 2004/0091985 to Metcalf et al., which isincorporated herein by reference. A phosphite dehydrogenase enzyme may(or may not) be a NAD-dependent enzyme with high specificity forphosphite as a substrate (e.g., Km ˜50 μM) and/or with a molecularweight of about 36 kilodaltons. The dehydrogenase enzyme may, but is notrequired to, act as a homodimer, and/or have an optimum activity at 35°C. and/or a pH of about 7.25-7.75.

Hypophosphite dehydrogenase—an enzyme that catalyzes oxidation ofhypophosphite to phosphite. The enzyme may, for example, be a bacterialenzyme, such as HtxA from Pseudomonas stutzeri WM 88 (SEQ ID NO:20;GenBank AAC71711.1) or Alcaligenes faecalis (GenBank AAT12775.1).

An HtxA polypeptide may, but is not required to, be a Fe-dependentenzyme with high specificity for hypophosphite as a substrate (e.g., Km˜0.54-0.62 mM) and/or with a molecular weight of about 32 kilodaltons.The HtxA polypeptide may, but is not required to, act as a homodimer,and/or to have an optimum activity at 27° C. and/or a pH of about 7.0.

ptxD or htxA coding region—a sequence encoding a PtxD polypeptide (i.e.,a phosphite dehydrogenase enzyme) or an HtxA polypeptide (i.e., ahypophosphite dehydrogenase enzyme), respectively. An exemplary ptxDcoding region is provided by ptxD of Pseudomonas stutzeri (SEQ ID NO:21;GenBank AF061070.1). In other examples, a ptxD-like coding region withat least 80% or 90% sequence identity to SEQ ID NO:21 may be utilized.In other examples, a coding region that encodes a polypeptide with atleast 50%, 60%, 80%, 90%, 95% or complete identity to one or more of thepolypeptides of SEQ ID NOS:1-14 may be utilized.

II. GENERATION OF TRANSGENIC PLANTS AND FUNGI

The present disclosure provides methods of making transgenic plants andtransgenic fungi that have a modified metabolism of phosphorus. Themethods may be used to create, as a primary goal, transgenic plantsand/or fungi (or at least one plant or fungal cell) carrying a nucleicacid construct encoding an enzyme of phosphorus oxidation, such as forbetter growth on a phosphite and/or hypophosphite fertilizer inagriculture. Alternatively, or in addition, the methods may be used tocreate, as a primary goal, transgenic plants and/or fungi carrying aconstruct including another gene of interest, with the construct alsoincluding a gene encoding an enzyme of phosphorus oxidation acting as aselectable marker to facilitate identification and/or isolation of thetransgenic plants or fungi. The method steps disclosed in this sectionand elsewhere in the present disclosure may be performed in any suitablecombination, in any suitable order, and repeated any suitable number oftimes.

FIG. 1 shows a schematic flowchart of an exemplary method 20 of (i)making a transgenic plant (and/or fungus) that metabolizes at least onereduced form of phosphorus (“RP”) to phosphate and/or (ii) using, as aselectable marker, a nucleic acid that confers a capability tometabolize a reduced form of phosphorus to phosphate.

At least one construct (or nucleic acid) may be obtained, as indicatedat 22. The at least one construct 23 may include at least one first gene24, which may be at least one chimeric gene encoding at least one enzyme(“RP-OxRe”), such as a phosphite dehydrogenase, that catalyzes oxidationof a reduced form of phosphorus, such as oxidation of phosphite tophosphate. Construct 23 also may include at least one second gene 26(“Gene2”), which also may (or may not) be a chimeric gene. In someembodiments, the at least one first gene may be a pair of genes encodingat least two distinct polypeptides that each catalyze oxidation of atleast one reduced form of phosphorus. The at least two polypeptides mayact to oxidize phosphorus substrates in series (e.g., catalyzingoxidation of hypophosphite to phosphite with a first polypeptide andthen catalyzing oxidation of phosphite to phosphate with a secondpolypeptide). In some examples, the at least one second gene may includea selectable marker for use in plants and/or fungi and/or may include agene(s) of primary interest, among others. First gene 24 and second gene26 may be linked, such as being present in the same polynucleotide, ormay be present on respective discrete polynucleotides. Each gene may beconstructed, at least in part, outside of plants, such as in vitroand/or in a microorganism (e.g., bacteria, yeast, etc.). Furthermore,each gene may be capable of expression in plants, fungi, or both thatcontain the gene.

The at least one gene (24 and/or 26) may be introduced into at least onerecipient plant 28 (or fungus), plant or fungal tissue, and/or plant orfungal cell, indicated at 30. The at least one plant, tissue, or cell,prior to introduction of the at least one gene, may at leastsubstantially require phosphate as an external source of phosphorus forgrowth. In other words, the plant, tissue, or cell may be at leastsubstantially unable to metabolize directly a reduced form of phosphorus(such as phosphite) as a nutrient.

Introduction of the at least one gene may be performed by contacting (a)the at least one plant/fungus, tissue, and/or cell and (b) a composition(a modifying agent) that includes a nucleic acid comprising the at leastone gene, under conditions that encourage introduction of the nucleicacid into the plant, tissue, and/or cell. The step of contacting may beperformed by any mechanism that creates contact between the at least oneplant/fungus, tissue, and/or cell and the composition. The compositionmay, for example, include one or more polynucleotides containing the atleast one gene, with the polynucleotides in and/or on a carrier.Exemplary carriers that may be suitable include biological cells (e.g.,bacterial cells), plant viruses, inert particles, lipids (in micellesand/or liposomes), and/or the like. Exemplary contact created with acomposition including the gene may include contacting a plant, planttissue, or plant cells with a bacterium (e.g., an Agrobacterium species,such as Agrobacterium tumefaciens or Agrobacterium rhizogenes) carryingthe at least one gene, or with one or more projectiles carrying the atleast one gene (e.g., particles coated with a polynucleotide includingthe at least one gene and fired at the plant, tissue, or cell from agene gun). More generally, introducing the at least one gene may beperformed on a plant/fungus, plant or fungal tissue, and/or plant orfungal cells by infection, injection, particle bombardment,electroporation, cell fusion, lipofection, calcium-phosphate mediatedtransfection, any combination thereof, or the like.

Transgenic candidates 34 (also termed transformation candidates) may begenerated, indicated at 36, by and/or after creating contact between theplant, tissue, and/or cells and the composition. The transgeniccandidates may be the plant/fungus, tissue, and/or cells used forcontacting, or may be derived from any later generation (i.e., progenyor division products) of the plant/fungus, tissue, and/or cells. In anyevent, the transgenic candidates may be seeds, plants, tissues,explants, isolated cells, cell colonies/aggregates, and/or the like.

Selection for growth (i.e., a growth advantage) of transgenic candidates34 in a selective medium 37 may be performed, indicated at 38.Candidates 34 that possess a growth advantage on the selective medium,such as transgenic plants 40, generally are substantially larger thanthe other candidates. In other examples, the selection may be performedwith transformed plant (or fungal) cells and may include culturing theplant (or fungal) cells in a selective medium. In these cases, culturingthe cells may permit selection and/or isolation of one or more coloniesof cells formed by the step of culturing. The colonies may be expressingan enzyme, such as a phosphite dehydrogenase, that oxidizes a reducedform of phosphorus, as evidenced by formation of the colony in themedium.

Any suitable selective medium 37 may be utilized according to aselectable marker provided by the at least one first gene and/or aselectable marker (second) gene that was introduced. For example, theselective medium may include a reduced form of phosphorus, such ashypophosphite and/or phosphite. The reduced form of phosphorus may be aprimary external source of phosphorus and/or may be at leastsubstantially the only phosphorus present in the medium, which meansthat the medium is at least substantially without phosphate (i.e., a lowphosphate or no phosphate medium). Alternatively, or in addition, theselective medium may include another selective agent, such as hygromycinor phosphinothricin, if the selection for growth is based on second gene26 (e.g., hph or bar) introduced into the plant/fungus, tissue, and/orcell. If the selection is based on second gene 26, additional tests(e.g., growth in phosphite-containing medium, PCR, Southern blot, etc.)may be performed to test for introduction of the at least one first geneencoding at least one RP-oxidoreductase. In any event, the medium mayinclude or be predominantly liquid and may (or may not) include a matrixor substrate, such as a gel (e.g., agar, agarose, gelatin, etc.) orsoil, among others.

Selection for growth may be performed in any suitable vessel 42 (and/orcontainer) or may be performed without a vessel or container, such as ina field. Exemplary vessels that may be suitable are covered or uncoveredand include single- or multi-well plates or dishes (e.g., Petri dishes),pots, trays, boxes, etc.

Transgenic plant 40 may be isolated, indicated at 44. Plant 40 may havea growth advantage conferred by nucleic acid 23 for growth on a reducedform of phosphorus, relative to a non-transgenic variety of the plant(e.g., plant 28) from which transgenic plant 40 was derived. Stateddifferently nucleic acid 23 may confer a capability to metabolize thereduced form of phosphorus as a nutrient. In some embodiments,transgenic plant 40 may be regenerated from transformed plant cells ortissue. For example, at least a portion of a colony of cells produced bycultivating the plant cells in a selective (e.g., phosphite) medium maybe utilized to regenerate the transgenic plant. Further aspects ofgenerating transgenic plants and fungi are described elsewhere in thepresent disclosure, such as in the Examples of Section IV.

FIG. 2 shows a schematic representation of nucleic acid 23 for use inmethod 20 (FIG. 1). Gene 24 may be termed an RP-OxRe gene 46 thatexpresses, indicated at 48, a reduced phosphorus oxidoreductase 50(e.g., a phosphite dehydrogenase). Gene 24 includes a coding region 52that encodes the oxidoreductase. Gene 24 also may include atranscription promoter 54 operatively linked to coding region 52, and atranscription terminator 56 operatively linked to coding region 52.

Promoter 54 and terminator 56 may be functional in plants and/or fungi.Accordingly, the promoter and/or the terminator may originate from aplant or fungus, or a virus or a bacteria that infects plants or fungi,among others. Exemplary promoters that may be suitable for use in plantsinclude the 35S promoter of Cauliflower Mosaic Virus. Other promotersthat may be suitable for use in plants include a PLDZ2 promoter from thePhospholipase DZ2 (PLDZ2) gene (Gene model AT3G05630.1; TAiR accessionGene:2078036) of Arabidopsis thaliana, which is inducible underconditions of low phosphate availability to the plant (Cruz-Ramirez etal., PNAS 2006, 103:6765-6770, the disclosure of which is incorporatedherein by reference). Alternatively, or in addition, the promoter may bea root-specific promoter, such as the Arabidopsis Pht1;2 phosphatetransporter gene (NCBI NM_(—)123703.1; GeneID:834355) or the promoter ofthe MtPT1 gene or MtPT2 gene (GenBank: AF000354.1 and AF000355.1) ofMedicago truncatula (Xiao, et al, Plant Biology, 2006, 8:439-449, thedisclosure of which is incorporated herein by reference).

Furthermore, first gene 24 may include transcribed but untranslatedregions, such as a 5′ leader sequence and/or 5′ untranslated region 58,a 3′ untranslated region 60, and/or one or more introns 62. First gene24 may be provided by nucleic acid 23 that includes any other suitablesequences, such as at least one second gene 26, replication controlsequences for replication in bacteria or another non-plant species, aselectable marker for another species (e.g., bacteria), or anycombination thereof, among others. In some embodiments, nucleic acid 23may be any combination of linear or circular (i.e., a closed loop), atleast mostly double-stranded or at least mostly single-stranded, and DNAor RNA.

FIG. 3 shows a proposed mechanism 70 for oxidation of hypophosphite tophosphate in bacteria catalyzed by enzymes expressed from ptxD and htxAgenes. The proposed mechanism presented here is for illustrationpurposes only, and is not intended to limit the definition of any of thecomponents shown, such as ptxD or htxA genes or PtxD or HtxApolypeptides, or limit the scope of the invention.

Mechanism 70 shows a hypophosphite ion 72 may be oxidized to a phosphiteion 74 by the action of an HtxA polypeptide 76(hypophosphite:2-oxoglutarate dioxygenase) encoded by an htxA gene. HtxApolypeptide 76 may use Fe²⁺ 78 as a cofactor and 2-oxoglutarate 80 as anelectron donor. In addition, enzyme 76 may convert 2-oxoglutarate 80 tosuccinate 82, and molecular oxygen 84 to carbon dioxide 86.

Phosphite ion 74, in turn, may be oxidized to a phosphate ion 88 by theaction of a PtxD polypeptide 90. Polypeptide 90 may use NAD+ 92 as anelectron acceptor that is reduced to NADH 94.

III. USE OF TRANSGENIC PLANTS AND FUNGI

The transgenic plants disclosed herein may be used for any suitablepurpose. Exemplary purposes include production of a commercial product(e.g., food, wood, pharmaceuticals, dyes, oils, lubricants, inks,rubber, cotton, fibers, biofuels, etc.), and/or water remediation. Waterremediation, as used herein, generally includes any removal of pollutionor at least one contaminant from a body of water and/or from soil thathas contacted contaminated water.

A method of water remediation is provided. Any transgenic plant, fungi,or both disclosed herein may be used in the method. The method stepsdisclosed in the section and elsewhere in the present disclosure may beperformed in any suitable order, in any suitable combination, with eachstep performed any suitable number of times.

One or more transgenic plants may be obtained. The transgenic plants mayhave been transformed, in the current generation or in any proceedinggeneration, with a construct that confers a capability of oxidizing atleast one reduced form of phosphorus.

Obtaining the one or more transgenic plants may include any suitableprocedures. For example, the step of obtaining may include introducinginto the current generation or, more typically, an earlier generation ofthe transgenic plants, one or more constructs encoding one or morepolypeptides that oxidize a reduced form of phosphorus to phosphate.

The one or more transgenic plants may be contacted with water to beremediated. Contacting plants to with water may include any combinationof bringing the water to plants, bringing the plants to water, andgerminating seeds for the plants in contact with the water. The watermay be substantially stationary or may be flowing with respect to theplants. In some embodiments, the step of contacting may includecontacting the plants with an industrial and/or municipal effluent.

IV. EXAMPLES

The following examples describe selected aspects and embodiments of thepresent disclosure, such as exemplary methods of making transgenicplants (including algae) and transgenic fungi that metabolize phosphiteas a source of phosphorus, exemplary transgenic plants and transgenicfungi, and exemplary methods of using a gene encoding a phosphitedehydrogenase enzyme as a selectable marker for selection of transgenicplants and transgenic fungi. The examples are presented for illustrationonly and are not intended to define or limit the scope of the presentdisclosure.

Example 1 Exemplary Generation of Transgenic Plants Expressing aBacterial Phosphite Dehydrogenase Enzyme

This example describes an exemplary method of generating transgenicplants with modified phosphorus metabolism; see FIGS. 4-6.

FIG. 4 shows an exemplary nucleic acid, a chimeric gene 100, constructedfor use in generating a transgenic plant that metabolizes phosphite tophosphate, to permit growth on phosphite in the absence of phosphate.The gene was constructed using the Gateway® system (Gateway® Technology,2003, Invitrogen) as described in the following paragraphs.

Gene 100 includes a 35S promoter sequence 102 from Cauliflower MosaicVirus (CaMV) operatively linked to a coding sequence 104 (SEQ ID NO:21)from ptxD of Pseudomonas stutzeri WM88. Expression of gene 100,indicated at 106, to produce the PtxD polypeptide (a phosphitedehydrogenase enzyme) is thus controlled/driven by 35S promoter 102.Gene 100 optionally may include a termination sequence 107, such as a35S terminator from CaMV, disposed downstream of and operatively linkedto the coding sequence (and promoter sequence). The gene further mayinclude a 5′ untranslated sequence disposed between the promotersequence and the ptxD coding sequence, and/or a 3′ untranslated sequencedisposed between the ptxD coding sequence and the termination sequence.Furthermore, the gene may include an intron that is transcribed alongwith ptxD coding sequence and that is removed from the transcript bypost-transcriptional splicing.

FIG. 5 shows a schematic diagram of a portion of a strategy used tocreate gene 100 of FIG. 4. A forward primer 108 (SEQ ID NO:22) and areverse primer 110 (SEQ ID NO:23) were synthesized. Each primer has ahybridization region 112, 114 that hybridizes, indicated at 116, 118 inthe lower part of the figure, in either a forward or reverse orientationto the ends of ptxD coding region 104. Each primer has an attB site 120,122 (attB1 or attB2) positioned 5-prime to hybridization region 112 or114. The primers were utilized to amplify coding sequence 104 from aplasmid (pWM302) using the polymerase chain reaction, to create a ptxDamplified product. A construct of the expected size, about 1000 basepairs, was generated, as detected by gel electrophoresis and staining ofthe amplified product. The primers alternatively may be designed toamplify additional untranslated sequences from upstream and/ordownstream of the ptxD coding sequence.

The ptxD amplified product next was incorporated into a plasmid vectorusing site-specific recombination provided by the Gateway® system. Theamplified product was recombined with plasmid pDONR221, via the attP1and attP2 sites of pDONR 221 and the attB1 and attB2 sites of theamplified product, to create a ptxD derivative of pDONR221, “initialclone” pDONR221. The initial clone has the full-length ptxD codingsequence opposingly flanked by attL1 and attL2 sites.

The ptxD sequence of the initial clone then was moved into an acceptorvector by further site-specific recombination to produce an expressionconstruct, pB7WG2D-ptxD. The acceptor vector was pB7WG2D.1, whichincludes, in order around the vector, (1) a 35S promoter, (2) attR1 andattR2 sites disposed downstream of the 35S promoter, (3) a 35Sterminator, (4) a “bar” gene (confers phosphinothricin resistance) as aselectable marker in plants, (5) a gene, SmSp^(R), as a selectablemarker in bacteria, particularly Agrobacterium (confers spectinomycin(Sp) and streptomycin (Sm) resistance), and (6) an EgfpER gene.Gateway®-system directed recombination formed expression clone(pB7WG2D-ptxD) including gene 100 (see FIG. 4), bar, SmSp^(R), andEgfpER.

The expression construct was used to transform electrocompetentAgrobacterium tumefaciens by electroporation. A transformedAgrobacterium clone carrying the expression construct was selected forsubculture.

The transformed Agrobacterium clone was used to transform Arabidopsisthaliana (ecotype Col-0) (generally described herein as “wild-type”(WT)) using a modified floral dip method. Transformed T0 progeny wereselected using phosphinothricin resistance. In particular, screening wasperformed with MS 0.1× media containing phosphinothricin (20 mg/L).Twenty-eight resistant lines were identified through PCR amplificationof the ptxD gene. Each resistant line was analyzed via T1 progeny usingMS 0.1× media containing phosphinothricin (20 mg/L) to look for 3:1(resistant:sensitive) segregation of the T1 progeny, to identify plantsthat showed Mendelian transmission of the ptxD gene. Ten homozygous ptxDtransgenic plants were established from T2 progeny of T1 progenyexhibiting 3:1 transmission.

The ptxD transgenic plants were tested for their ability to grow inmedia containing only phosphite (e.g., about 0.1 to 5 mM) as an externalsource of phosphorus. Control plants showed no substantial growth inthis media (i.e., showed growth limited to the internal phosphorusreserves accumulated in the seed), whereas the transgenic plants grewefficiently, thereby demonstrating that the transgenic plants are ableto metabolize a reduced form of phosphorus (phosphite) as a source ofphosphorus.

The ptxD expression construct also was used to provide a selectablemarker for selection of transgenic plants with modified phosphorusmetabolism. Wild-type Col-0 plants were transformed using Agrobacteriumcontaining the ptxD expression construct. T0 progeny (seeds) were platedon a medium with phosphite (5 mM) as the source of phosphorus. FIG. 6shows exemplary data for growth of the T0 progeny, relative to wild typeplants, on the phosphite medium. Transgenic plants 130 (circled in theright panel) have a substantial growth advantage relative to wild typeplants 132 (left panel) and relative to other T0 progeny 134 thatapparently were not transformed with the expression construct and/orthat did not efficiently express the PtxD polypeptide from theintroduced construct.

Further aspects of generating transgenic plants with modified phosphorusmetabolism are described in U.S. Provisional Patent Application Ser. No.61/199,784, filed Nov. 19, 2008, which is incorporated herein byreference.

Example 2 Characterization of Arabidopsis Plants Expressing PtxD

This example presents an investigation of the growth characteristics ofthe parental (“wild-type” (WT) or control) Arabidopsis line, Col-0, andtwo of the transgenic Arabidopsis lines described in Example 1 andcomprising the ptxD expression construct of Example 1; see FIGS. 7-12.

Two transgenic Arabidopsis lines, dubbed PTXD-3 and PTXD-5, wereprepared and isolated as described in Example 1. Each line is homozygousfor the ptxD expression construct of Example 1.

The parental line and the PTXD-3 and PTXD-5 transgenic lines were testedfor the ability to grow on a liquid medium, with or without inorganicphosphate (Pi) as the source of phosphorus. Seeds from the parental andtransgenic lines were germinated in liquid media and tested for growth.In the absence of phosphate (and phosphite), neither the parental linenor the transgenic lines showed significant growth beyond germination.(Each line exhibited paltry growth for a short time, which apparentlywas permitted by phosphate stores in the seeds, which are quicklydepleted from the seeds.) In contrast, both the parental (WT) line andthe transgenic lines grew efficiently in the presence of 50, 100, and1000 μM phosphate.

FIG. 7 shows photographs of data obtained from tests of the growth ofthe parental (WT) line and the transgenic PTXD-3 and PTXD-5 lines on aliquid growth medium, with or without phosphite (Phi) or phosphate (Pi)as the source of phosphorus. In FIG. 7, the absence or presence ofsustained plant growth (beyond the germination stage) is identified witha minus (−) or a plus (+) symbol, respectively. Both the parental lineand the transgenic lines grew efficiently in the presence of 50 μMinorganic phosphate (bottom row). Also, neither the parental line northe transgenic lines showed detectable growth in the absence of bothphosphate and phosphite. However, both transgenic lines, but not theparental line, grew efficiently in the presence of 50, 100, and 1000 μMinorganic phosphite as phosphorus source. Therefore, the transgeniclines acquired the ability to metabolize phosphite as a phosphorussource to support plant growth.

FIG. 8 shows a bar graph of data obtained from tests of the ability ofthe wild-type and transgenic Arabidopsis lines of FIG. 7 to reduce theamount of total phosphorus in a growth medium containing differentconcentrations of phosphite (50, 100, and 1000 μM) as the source ofphosphorus.

Wild type and the two transgenic Arabidopsis lines, PTXD-3 and PTXD-5,were germinated and cultivated in one-liter plastic containers with 0.1×Murashige and Skoog liquid medium lacking phosphate and supplementedwith either 50, 100 or 1000 micromolar phosphorous acid (H₃PO₃). Onehundred plants per plastic container were allowed to grow for 45 days inthe plastic container in a growth chamber with a 16:8 light:dark cyclefor each 24-hour period. The plants were covered to avoid moisture loss.A double layer of plastic mesh was placed where seeds were sown togerminate on top of liquid media in each plastic container.

After 45 days of growth the total phosphorus content was determined inthe liquid media, after removing the plants, using a vanadium-molybdatemethod. Briefly, 5 mL of liquid media from each sample was digested withnitric acid:perchloric acid (HNO₃:HClO₄; 5:1). Then, the phosphoruscontent was determined with a colorimetric method based on the additionof a solution of ammonium molybdate (20 mM) and ammonium metavanadate(10 mM) in 70% perchloric acid. After a 20-minute incubation at roomtemperature, the absorbance at 400 nm was measured with aspectrophotometer.

In FIG. 8, the first three bars labeled as “initial” represent theinitial concentration of total phosphorus in the media without plants.The sets of bars labeled as WT (Col-0), PTXD-3, and PTXD-5 represent thetotal phosphorus content in the media (initially 50 μM, 100 μM, or 1000μM phosphite) after 45 days of incubation in the presence of thecorresponding Arabidopsis lines. The transgenic plants (PTXD-3 andPTXD-5), but not the wild-type plants, diminished the phosphorus contentin the media by more than 50%. The decrease in phosphorus content, whichin this case represents a removal of phosphite from the media, is due tothe uptake of phosphite by the plants. The transgenic lines have a highcapacity to remove phosphite from the media because they are able toconvert it into phosphate, which sustains plant growth. This ability toremove phosphite from an aqueous medium may be exploited to removephosphite from waste water, such as effluents produced by CD/DVDfactories.

FIG. 9 shows a schematic representation of the distribution of parental(WT) and ptxD transgenic (PTXD) Arabidopsis plants used for theexperiments of FIGS. 10 and 11.

FIGS. 10 and 11 show photographs of parental and ptxD transgenic plantsdistributed according to FIG. 9 and tested for growth on a substratecontaining added phosphate (Pi) (FIG. 10) or phosphite (Phi) (FIG. 11)as the source of phosphorus. The presence or absence of sustained growth(beyond the germination stage) is indicated by a plus (+) or a minus (−)symbol, respectively. FIG. 10 shows similar growth of wild-type andtransgenic plants on phosphate. In contrast, FIG. 11 shows that only thetransgenic plants were capable of sustained growth on phosphite. Theplants here and in FIG. 11 were grown in a sand:vermiculite mixture(1:1) and received water and nutrient solutions (lacking any otherphosphorus source except as previously indicated) periodically.

FIG. 12 shows a bar graph of data obtained from tests of the ability ofthe Arabidopsis plant lines of FIG. 7 to increase in weight whencultivated in the presence of various sources of phosphorus. The dryweight of three plants cultivated in sand:vermiculite (1:1) as substrateis plotted in the figure with respect to each particular plant line andsource(s) of phosphorus. Wild type plants did not grow substantiallywith phosphite as the source of phosphorus, while the transgenic linesgrew similarly or better on phosphite (Phi) relative to phosphate (Pi).

Example 3 Transgenic Tobacco Plants Expressing PtxD

This example describes the creation and characterization of transgenicNicotiana tabacum (tobacco) comprising the ptxD expression construct ofExample 1; see FIG. 13.

Nicotiana tabacum was transformed with the expression constructdescribed in Example 1. In particular, tobacco leaf explants wereco-cultivated with an Agrobacterium strain harboring a 35S::PtxDconstruct (Example 1) within its T-DNA. Leaf discs were allowed toregenerate in MS media containing 1 mM phosphite as the only phosphorussource. Plants regenerated from these leaf discs on phosphite-containingmedia were transferred to soil and allowed to set seed under greenhouseconditions.

FIG. 13 shows photographs of T2 transgenic tobacco seeds, homozygous forthe 35S::PtxD gene, and control tobacco seedlings taken 25 days aftergermination in MS media containing either phosphate (1 mM Pi) orphosphite (1 mM Phi) as the only phosphorus source. The presence orabsence of growth (after depletion of seed-furnished phosphorus) isindicated by a plus (+) or a minus (−) symbol, respectively. It can beseen that the control seedlings germinated but were unable to sustainnormal growth in phosphite-containing media, compared to when phosphateis supplied as a phosphorus source. In contrast, tobacco plants fromeach transgenic line showed sustained growth in the presence ofphosphite or phosphate as the source of phosphorus. These experimentsdemonstrate the ability to modify phosphorus metabolism in tobacco.

FIG. 14 shows photographs of additional growth experiments performedwith the control and transgenic tobacco lines of FIG. 13. Seedlings weregerminated and maintained in MS media supplemented with 1 mM phosphiteas the only phosphorus source for 25 days. The seedlings then weretransferred to tissue culture flasks containing MS with 1 mM phosphiteas the only phosphorus source and were allowed to grow for 25 additionaldays in a plant growth chamber at 23° C., with a photoperiod of 18 hlight, followed by 6 h darkness for each 24-hour period. It can beobserved that the PTXD transgenic plants are able to sustain rapidgrowth in media containing phosphite as the only phosphorus source,whereas the control plant is unable to use phosphite for its growth anddevelopment.

Example 4 Transgenic Algae with Modified Phosphorus Metabolism

This example describes a method of creating a transgenic line of algaeexpressing a phosphite dehydrogenase enzyme that enables growth of thealgae on phosphite as a source of phosphorus.

Photosynthetic algae have been adapted transgenically for manyapplications, such as production of biofuels, pharmaceuticals, antigens,and the like. The algae can be cultured in large fermentation tanks thatincorporate a light system to support photosynthesis and promote growth.Generally, the fermentation tanks must be protected from contaminationwith undesirable algae (or other organisms). Toward this end, the algaeare grown under artificial light rather than sunlight, to reduce therisk of contamination. Accordingly, growth of the algae with exposure tosunlight in open tanks or fields (e.g., in ponds), which would be muchcheaper, is not feasible currently because of the high risk ofcontamination.

The present disclosure enables the use of sunlight and open fields forgrowth of target algae by modifying the target algae for growth onphosphite as a source of phosphorus. The modified target algae would becapable of thriving in a medium containing phosphite and lackingphosphate, which would not support growth of unwanted (contaminant)algae because they would require phosphate. Accordingly, contaminationby the unwanted algae would be reduced or eliminated, permitting thetarget algae to be cultured at a lower cost in an open tank or fieldwith photosynthesis driven by sunlight.

An expression construct for transformation of an algae species, such asChlamydomonas reinhardtii, is generated. The construct can express anysuitable phosphite dehydrogenase (and, optionally, a hypophosphitedehydrogenase, too). In the present illustration, the constructexpresses PtxD from the ptxD coding sequence. The construct utilizes ahybrid promoter sequence to drive expression while avoiding genesilencing: the HSP70A promoter is fused upstream of the RBCS2 promoter(each promoter is provided by C. reinhardtii) (Schroda et al, 2000,Plant J. 21: 121-131). The hybrid promoter sequence drives expression ofthe first intron of RBS2 of C. reinhardtii, which is fused to the codingsequence of the ptxD gene (Pseudomonas stutzeri), which, in turn, isfused to the transcription termination sequence of the RBS2 gene. Toenhance expression of PtxD polypeptide from the construct, the ptxDcoding sequence may be modified to have a G or C in the third positionof codons that permit this change (via degeneracy of the genetic code),to optimize codon usage for translation in C. reinhardtii.

The ptxD expression construct may be provided as a plasmid containing anorigin of replication functional in E. coli, a selectable marker for E.coli (e.g., an ampicillin-resistance gene), and a selectable markerfunctional in C. reinhardtii, among others. An exemplary selectablemarker for C. reinhardtii encodes a zeomycin binding protein thatconfers resistance to zeomycin and phleomycin (Lumbreras et al., 1998,Plant J. 14: 441-447).

The ptxD expression construct is introduced into C. reinhardtii by anysuitable mechanism, such as particle bombardment (Debuchy et al., 1989,EMBO J. 8: 2803-2809) or with the aid of glass beads (Kindle et al.,1991, PNAS 88: 1721-1725), among others.

Transformation of C. reinhardtii with glass beads can be carried out asdescribed by Kindle (1990, PNAS 87: 1228-1232). Cell walls are removedfrom C. reinhardtii cells by incubating them in undiluted autolysin for30-60 min at room temperature. The effectiveness of treatment ismonitored by sensitivity to 0.004% Nonidet P-40 detergent (Sigma). Cellsare harvested from autolysin by centrifugation, resuspended in liquidmedium, and transformed immediately to avoid cell-wall regeneration.Glass beads (0.45-0.52 mm) are washed with concentrated sulfuric acid,then rinsed thoroughly with distilled water, dried, and sterilized bybaking at 250° C. for 2-3 h. Glass beads (300 mg) are added to 0.4 mL ofcells, 2 micrograms of plasmid DNA is added, and cells agitated at topspeed on a Fisher Vortex Genie II mixer in 15-mL conical disposablepolypropylene centrifuge tubes. The beads are allowed to settle, andcells are spread on selective agar plates with a glass spreader. Fordirect selection of zeomycin-resistant transformants, cells are agitatedwith glass beads and DNA, diluted in 20 mL TAP liquid medium and left toexpress the ble gene by incubating at 25° C. in the light (80 μE m⁻²s⁻¹) for 15-18 h with gentle shaking. Cells are then pelleted bycentrifugation, resuspended in 5 mL of TAP containing 0.5% molten agar,and poured onto the surface of a TAP/2% agar plate containing zeomycinat 20 mg/mL.

Zeomycin-resistant colonies are then spread in TAP media lacking anysource of phosphate, but supplemented with 1 mM phosphite as aphosphorus source. Plates are incubated for 18 to 24 h at 25° C. inlight and colonies that grow are able to use phosphite as a phosphorussource.

Example 5 Transgenic Trichoderma Expressing a Phosphite Dehydrogenase

This example describes a method of creating a fungus of the genusTrichoderma modified to express a phosphite dehydrogenase enzyme, torender the fungus capable of growing on phosphite as a source ofphosphorus.

A. Introduction

Trichoderma species are free-living fungi that are common in soil androot ecosystems. Recent discoveries show that they behave as avirulentplant symbionts, as well as being parasites of phytophatogenic fungi.Some strains establish robust and long-lasting colonization of rootsurfaces and penetrate into the epidermis. As ubiquitous soilinhabitants and rhizosphere-competent fungi, Trichoderma species havebeen used successfully as biological control agents for the managementof plant pathogens. Several mechanisms of biocontrol have been proposedfor Trichoderma, including competition, mycoparasitism, and theinduction of plant defense responses due to colonization of plant rootintercellular spaces (Howell, 2003; Yedidia et al., 1999). Rootcolonization by Trichoderma species also frequently enhances root growthand development, crop productivity, resistance to abiotic stresses, andthe uptake and use of nutrients.

Trichoderma species may be modified to express PtxD or an ortholog orderivative thereof, to render the Trichoderma capable of growth onphosphite. Optionally, the Trichoderma also may be modified to express ahypophosphite dehydrogenase (e.g., HtxA). In any event, these transgenicTrichoderma may be put to various uses. For example, they may be usedfor bioremediation purposes, such as to eliminate phosphite (and/orhypophosphite) from waste water discharge of the CD and DVD industry.The transgenic Trichoderma can be utilized for bioremediation alone, orin combination with a transgenic plant (e.g., Example 1). Use of acombined transgenic plant/fungal system for removal of phosphite (and/orhypophosphite) may be more efficient than the use of either alone.Alternatively, the transgenic Trichoderma can be associated with plantsto protect them from pathogen fungi. In this case, the plants may benon-transgenic such that they require phosphate as a source ofphosphorus, or may be transgenic plants that can grow on phosphite as asource of phosphorus (e.g., Example 1). In any event, the transgenicTrichoderma may function as a powerful fungicide, since both theTrichoderma itself, and its utilization of phosphite may protect theplants.

B. Protocol

Transformation of Trichoderma atroviride (IMI 206040) protoplasts iscarried out using methods known to the art, such as the PEG-CaCl₂ method(Herrera-Estrella et al., 1990; Baek & Kenerley, 1998), biolistics(Lorito et al., 1993), or electroporation (Goldman et al., 1990), amongothers. The transforming DNA is a plasmid or a PCR product carrying agene encoding a phosphite dehydrogenase enzyme (e.g., PtxD) undercontrol of the Trichoderma reesei pki promoter or the Aspergillusnidulans trpC promoter, and the T. atroviride blu17 or the A. nidulanstrpC terminator. Plasmids are purified using the Qiagen Plasmid Midi Kitor cesium chloride gradients. For selection, 100, 200, and 500 μLaliquots are plated using an agar overlay containing 1.2 M sorbitol and200 mM H₃PO₃ as sole phosphorus source, immediately after treatment orafter a 2-4 hour incubation period of the protoplasts in 1.2 M sorbitol.After three to four days of incubation at 28° C., colonies capable ofgrowth on phosphite as the phosphorus source should appear on theplates. Transformants should appear only when transformed withconstructs carrying the ptxD coding sequences. Transformants aresubjected to three rounds of monosporic culture to obtain homokaryons.Alternatively, Trichoderma transformants may be obtained byco-transformation using an antibiotic resistance marker for selection(such as hph, which confers hygromycin resistance), in combination witha construct carrying the ptxD gene. Under the latter strategy,hygromycin-resistant transformants carrying the ptxD gene are firstselected, and strains capable of using phosphite as a phosphorus sourcecan be selected at a later stage as mentioned above, or are identifiedin a screen by testing expression of the ptxD gene.

Conidia of transformants carrying a phosphite-utilization cassette areproduced by solid or submerged fermentation processes known in the art(Cavalcante et al., 2008). The conidia may be applied to plants, seedstherefor, or to soil, among others. For example, the conidia may beapplied to seeds (e.g., with a latex sticker, such as Rhoplex B-15J),directly to plant roots as a spore suspension (e.g., with a sticker), orto soil in water as a spore suspension or in a wheat bran/peatpreparation mixture (0.5%, w/w), among others.

The following references are incorporated herein by reference:

-   Baek, J. M. & Kenerley, C. M. (1998). The arg2 gene of Trichoderma    virens: cloning and development of a homologous transformation    system. Fungal Genet. Biol. 23:34-44.-   Cavalcante, R. S., Lima, H. L. S., Pinto, G. A. S., Gava, C. A. T.,    and Rodrigues, S. (2008). Effect of Moisture on Trichoderma Conidia    Production on Corn and Wheat Bran by Solid State Fermentation. Food    Bioprocess. Technol. 1:100-104.-   Goldman, G. H., Van Montagu, M., and Herrera-Estrella, A. (1990).    Transformation of Trichoderma harzianum by high-voltage electric    pulse. Curr. Genet. 17:169-174.-   Herrera-Estrella, A., Goldman, G. H., and Van Montagu, M. (1990).    High efficiency transformation system for the biocontrol agents,    Trichoderma spp. Mol. Microbiol. 4:839-843.-   Howell, C. R. (2003) Mechanisms employed by Trichoderma species in    the biological control of plant diseases. Plant Dis. 87:4-10.-   Lorito, M., Hayes, C. K., Di Pietro, A., and Harman, G. E. (1993).    Biolistic transformation of Trichoderma harzianum and Gliocladium    virens using plasmid and genomic DNA. Curr. Genet. 24:349-356.-   Yedidia, I., Benhamou, N., and Chet, I. (1999). Induction of defense    responses in cucumber plants (Cucumis sativus L.) by the biocontrol    agent Trichoderma harzianum. Appl. Environ. Microbiol. 65:1061-1070.

Example 6 Mycorrhizae Formed with a Fungus Expressing a BacterialPhosphite Dehydrogenase Enzyme

This example describes a method of creating a mycorrhizal-type fungusmodified transgenically to express a phosphite dehydrogenase enzyme(and/or a bacterial hypophosphite dehydrogenase enzyme), which rendersthe transgenic fungus capable of growing on phosphite (and/orhypophosphite) as the source of phosphorus. A method is also disclosedof forming mycorrhizae by associating the transgenic fungus with aplant. Mycorrhizae formed with these transgenic fungi and the plant cansupply the plant with phosphate for growth. Accordingly, the plantitself would not need to be transgenic, since the mycorrhizae would doall the work of converting phosphite (and/or hypophosphite) intophosphate.

A. Introduction

Phosphorus (P) is an essential nutrient that can limit plantproductivity in natural and agricultural ecosystems. A plant can form anatural symbiotic relationship with a mycorrhizal fungus, which acts asan extension of the plant's root system to provide the plant withmineral nutrients, particularly phosphate, in exchange forcarbon-containing molecules derived from the plant's photosyntheticactivity (Smith and Read, 1997). Mycorrhizal fungi penetrate root cellsof the plant, with the plasma membranes of the fungi and plantestablishing a close association to form so-called arbuscularstructures. Mineral nutrients, particularly phosphate, can betransferred from fungal cells to plant cells in the arbuscularstructures. In addition to mineral nutrients, mycorrhizae can alsoimprove the ability of the plant to uptake water and can protect it fromheavy metals (Khan, A. G., 2006; Forbes et al., 1998).

Mycorrhizae have to compete with other microorganisms for phosphateavailability. Therefore, transgenic mycorrhizal strains that express agene encoding a phosphite dehydrogenase enzyme capable of convertingphosphite into phosphate can be used to supply plants with phosphate. Inthis case, the mycorrhizal fungus will convert phosphite into phosphate,which then may be transferred to the roots of non-transgenic plantsunable to metabolize phosphite. Alternatively, to make the system moreefficient, an association of transgenic mycorrhizal fungi and transgenicplants both expressing a gene encoding phosphite dehydrogenase can beused. The association of transgenic mycorrhizal fungi withnon-transgenic or transgenic plants can be used to enhance plantproductivity using fertilizers in which phosphate has been replaced byphosphite, or to bioremediate effluents from CD or DVD producingfactories or soils in which phosphite has been used as a fungicide(Ohtake, H., 1995)

B. Protocol

This example utilizes the ptxD coding sequence from Pseudomonasstutzeri. However, any suitable coding sequence for a phosphitedehydrogenase may be exploited.

A gene construct is created by placing the ptxD coding sequence undercontrol of the Aspergillus nidulans glyceraldehyde-3-phosphatedehydrogenase (gpd) promoter and the transcription terminator region ofthe A. nidulans tryptophan synthetase (trpC) gene. A selectable markersuch as the aph gene from E. coli, which confers resistance tohygromycin, or the ble gene, which confers resistance to phleomycin, isalso included in the transforming molecule (Barrett et al., 1990).

For transformation, protoplasts of a mycorrhizal fungus (e.g., Laccariabicolor, Cenococcum geophilum, Hebeloma cylindrosporium, Paxillusinvolotus, Gigaspora rosea, Glomus mosseae, Glomus aggregatum, Glomusintraradices, Pisolithus tinctorius, etc.) are obtained according to theprotocol of Barrett et al. (1990). To isolate protoplasts, mycelia arecollected and washed several times with sterile water and then treatedwith hydrolytic enzymes (a mixture of cellulase, chitinase, andproteases, with 5 to 10 mg/mL of each enzyme) in an osmotic solution(PDB; potato-dextrose-broth with 0.8 M mannitol or 0.6 M sucrose) todegrade the cell walls. The mycelia are incubated with the enzymes for 1to 3 hours at 32° C. with constant agitation (100 rpm). The protoplastsuspension is filtered and washed with the osmotic solution. Protoplastsare recovered by centrifugation for 10 min at 800 rpm and the protoplastpellet resuspended in PDB buffer and the number of protoplastsdetermined by counting under a microscope.

Protoplasts (1-3×10⁷ in 250 μL) are mixed with 5 to 20 micrograms of thegene construct and incubated in PEG transformation solution (25-60%polyethylene glycol 4000, 10-25 mM CaCl₂, 10 mM Tris-HCl, pH 7.5) for 45minutes at 4° C. One mL of additional PEG transformation solution isadded and incubation is continued at room temperature for 20 minutes.Protoplasts are allowed to regenerate cell walls in liquid media andtransformants are selected in solid media. The solid media (Potatodextrose agar) contains 100 μg/mL hygromycin or 100 μg/mL of phleomycin,depending on the selectable marker used for the transformation. Growingcolonies are transferred to solid media three times to isolate stablytransformed mycelia. The presence of the selectable marker as well asthe ptxD gene is confirmed by PCR. Once stable transformants areisolated, a 2 mm portion of mycelium is transferred to PDA media lackingphosphate and supplemented with 1 mM phosphite to identify colonies thatexpress the ptxD gene construct. Southern blot analysis is used toconfirm the presence of the corresponding genes.

To confirm that the transgenic fungus can provide phosphate to plants,soil is inoculated with mycelia of the ptxD-transformed fungus, andtobacco seed is germinated in the soil. The soil is fertilized with anormal concentration of nitrogen and potassium, with phosphite as thephosphorus source. Growth of tobacco plants from the seed in soilinoculated with the ptxD transgenic fungus is compared to growth incontrol soil that has not been inoculated.

The following references are incorporated herein by reference:

-   Barrett, V., Dixon, R. K., and Lemke, P. A. (1990) Genetic    transformation of a mycorrhizal fungus. Appl. Micobiol. Biotechnol.    33:313-316.-   Bills, S. N., Richter, D. L., and Podila G. K. (1995) Genetic    transformation of the ectomycorrhizal fungus Paxillus involutus by    particle bombardment. Mycological Research 99:557-561.-   Bills, S. N., Podila, G. K., and Hiremath, S. T. (1999) Genetic    engineering of the ectomycorrhizal fungus Laccaria bicolor for use    as a biological control agent. Mycologia 91: 237-242.-   Forbes, P. J., Millam, S., Hooker, J. E., and Harrier L. A. (1998)    Transformation of the arbuscular mycorrhiza Gigaspora rosea by    particle bombardment. Mycol. Res., 102:497-501.-   Hanif, M., Pardo, A. G., Gorier, M., and Raudaskoski, M. (2002)    T-DNA transfer and integration in the ectomycorrhizal fungus Suillus    bovinus using hygromycin B as a selectable marker. Curr. Genet.    41:183-188.-   Kemppainen, M., Circosta, A., Tagu, D., Martin, F., and    Pardo, A. G. (2005) Agrobacterium-mediated transformation of the    ectomycorrhizal symbiont Laccaria laccata S238N. Mycorrhiza.    16:19-22.-   Khan, A. G. (2006) Mycorrhizoremediation—an enhanced form of    phytoremediation. Journal of Zhejiang University Science B.    7:503-514.-   Marmeisse, R., Gay, G., Debaud, J-C., and Casselton, A. (1992)    Genetic transformation of the symbiotic basidiomycete fungus    Hebeloma cylindrosporum. Curr. Genet., 22:41-45.-   Ohtake, H., 1995. Applications of biotechnology to pollution    prevention. Bioremediation: the Tokyo '94 Workshop, OECD, Paris, pp.    409-417.-   Pardo, A., Hanif, M., Raudaskoski, M., and Golfer, M. (2002) Genetic    transformation of ectomycorrhizal fungi mediated by Agrobacterium    tumefaciens. Mycol. Res. 106: 132-137.-   Pardo, A. G., Kemppainen, M., Valdemoros, D., Duplessis, S., Martin,    F., and Tagu, D. (2005) T-DNA transfer from Agrobacterium    tumefaciens to the ectomycorrhizal fungus Pisolithus microcarpus.    Revista Argentina de Microbiologia. 37:69-72.-   Peng, M, Lemke, P. A., and Shaw J. J. (1993) Improved conditions for    protoplast formation and transformation of Pleurotus ostreatus.    Appl. Micobiol. Biotechnol. 40:101-106.-   Smith S. E., and Read D. J. (1997) Mycorrhizal Symbiosis. Academia    Press, San Diego, Calif. USA.

Example 7 Selected References

This example presents a set of references pertinent to aspects of thepresent disclosure. Each of the references is incorporated herein byreference for all purposes.

A. General Aspects of Phosphite in Plants

-   Ticconi, C. A., Delatorre, C. A., and Abel, S. (2001) Attenuation of    phosphate starvation responses by phosphite in Arabidopsis. Plant    Physiol. 127:963-972.-   Varadarajan, D. K., Karthikeyan, A. S., Matilda, P. D., and    Raghothama, K. G. (2002) Phosphite, an analogue of phosphate,    suppresses the coordinated expression of genes under phosphate    starvation. Plant Physiol. 129:1232-1240.-   Ouimette, D. G., and Coffey, M. D. (1989) Phosphonate levels in    Avocado (Persea amercana) seedlings and soil following treatment    with Fosetyl-Al or potassium phosphonate. Plant Dis. 73:212-215.-   Ouimette, D. G., and Coffey, M. D. (1990) Symplastic entry and    phloem translocation of phosphonate. Pestic. Biochem. Physiol.    38:18-25.-   Niere, J. O., DeAngelis, G., and Grant, B. R. (1994) The effect of    phosphonate on the acid-soluble phosphorus components in the genus    Phytophthora. Can. J. Microbiol. 140:1661-1670.-   Guest, D., and Grant, B. R. (1991) The complex action of    phosphonates as antifungal agents. Biol. Rev. 66:159-187.-   Förster H., Adaskaveg, J. E., Kim, D. H., and    Stanghellini, M. E. (1998) Effect of Phosphite on Tomato and Pepper    Plants and on Susceptibility of Pepper to Phytophthora Root and    Crown Rot in Hydroponic Culture. Plant Dis. 82:1165-1170.-   Carswell, C., Grant, B. R., Theodorou, M. E., Harris, J., Niere, J.    O., Plaxton, W. C. (1996) The Fungicide Phosphonate Disrupts the    Phosphate-Starvation Response in Brassica nigra Seedlings. Plant    Physiol. 110:105-110.-   Sukarno N., Smith, S. E., and Scott, E. S. (1993) The effect of    fungicides on vesiculararbuscular mycorrhizal symbiosis: I. The    effects on vesicular-arbuscular mycorrhizal fungi and plant growth.    New Phytol. 25:139-147.-   Ohtake, H. (1995). Applications of biotechnology to pollution    prevention. Bioremediation: the Tokyo '94 Workshop, OECD, Paris, pp.    409-417.-   Ohtake H., Wu, H., Imazu, K., Anbe, Y., Kato, J., and    Kuroda, A. (1996) Bacterial phosphonate degradation, phosphite    oxidation and polyphosphate accumulation. Resources, Conservation    and Recycling 18: 125-134.-   Albrigo, L. G. (1999) Effects of foliar applications of urea or    nutriphite on flowering and yields of Valencia orange trees. Proc.    Fla. State Hort. Soc. 112:1-4.-   Fenn, M, E., and Coffey, M. D. (1984) Studies on the In Vitro and In    Vivo Antifungal Activity of Fosetyl-Al and Phosphorous Acid.    Phytopathology. Vol. 74, No. 5.-   Schroetter, S., Angeles-Wedler, D., Kreuzig, R., and    Schnug, E. (2006) Effects of phosphite on phosphorus supply and    growth of corn (Zea mays). Landbauforschung Völkenrode 3/4 2006    (56):87-99.-   Rebollar-Alviter, A. L., Madden, L. V., and Ellis, M. A. (2005)    Efficacy of Azoxystrobin, Pyraclostrobin, Potassium Phosphite, and    Mafenoxam for Control of Strawberry Leather Rot Caused by    Phytophthora cactorum. Plant Health Progress.-   Wilcoxy, W. (2005) ProPhyt, Alliete, and Phosphorous Acid. The Lake    Erie Regional Grape Program.-   McDonald, A. E., Grant, B. R., and Plaxton, W. C. Phosphite    (Phosphorous Acid): Its Relevance in the Environment and Agriculture    and Influence on Plant Phosphate Starvation. J. Plant Nutr.    24:1505-1519.-   Niere, J. O., DeAngelis, G., and Grant, B. R. (1994) The effect of    phosphonate on the acid-soluble phosphorus components in the genus    Phytophthora. Microbiol. 140:1661-1670.-   Guest, D., and Grant, B. R. (1991) The complex action of    phosphonates as antifungal agents. Biol. Rev. 66:159-187.

B. Identification, Cloning, and Characterization of RP-Oxidoreductases

-   White, A. K., and Metcalf, W. W. (2007) Microbial Metabolism of    Reduced Phosphorus Compounds. Annu. Rev. Microbiol. 61:379-400.-   White, A. K., and Metcalf, W. W. (2002) Isolation and Biochemical    Characterization of Hypophosphite/2-Oxoglutarate Dioxygenase. A    Novel Phosphorus-Oxidizing Enzyme from Pseudomonas stutzeri WM88. J.    Biol. Chem. 277:38262-38271.-   Metcalf, W. W., and Wolfe, R. S. (1998) Molecular Genetic Analysis    of Phosphite and Hypophosphite Oxidation by Pseudomonas stutzeri    WM88. J. Bacteriol. 180:5547-5558.-   Garcia-Costas, A. M., White, A. K., and Metcalf, W. W. (2001)    Purification and Characterization of a Novel Phosphorus-oxidizing    Enzyme from Pseudomonas stutzeri WM88. J. Biol. Chem. 276:    17429-17436.-   Schink, B., Thiemann, V., Laue, H., and Friedrich, M. W. (2002)    Desulfotignum phosphitoxidans sp. nov., a new marine sulfate reducer    that oxidizes phosphite to phosphate. Arch. Microbiol. (2002)    177:381-391.

C. Transformation of Plants

-   Martinez-Trujillo, M. et al. (2004) Improving Transformation    Efficiency of Arabidopsis thaliana by Modifying the Floral Dip    Method. Plant Mol. Biol. Reporter. 22: 63-70.

Example 8 Selected Embodiments I

This example describes selected embodiments of the invention, presentedas a series of indexed paragraphs.

A. A transgenic plant capable of utilizing at least one reduced form ofphosphorus as a phosphorus fertilizer. The transgenic plant of thisparagraph may be further characterized as follows: (A1) wherein theplant expresses a bacterial coding sequence encoding an enzyme capableof oxidizing phosphite to phosphate, thereby permitting use of phosphiteas a phosphorus fertilizer (and a source of phosphorus); (A2) whereinthe bacterial coding sequence of A1 is ptxD from Pseudomonas stutzeri,Alcaligenes faecalis, or Xanthobacter flavus; (A3) wherein thetransgenic plant of A1 or A2 expresses htxA and ptxD coding sequences,thereby permitting use of hypophosphite and/or phosphite as a phosphorusfertilizer; (A4) wherein each or both of the bacterial coding sequencesof A3 is from Pseudomonas stutzeri, Alcaligenes faecalis, orXanthobacter flavus; (A5) wherein at least one of the bacterial codingsequence(s) of any of A1 through A4 is under control of a constitutivepromoter, a leaf-specific promoter, a tissue-specific promoter, aroot-specific promoter, a promoter inducible by low phosphate, or the35S promoter from the Cauliflower Mosaic Virus; or (A6) any combinationof A1 through A5.

B. The use of a transgenic plant capable of oxidizing hypophosphite tophosphate, and/or phosphite to phosphate, to eliminate hypophosphiteand/or phosphite from an industrial or municipal effluent.

C. The use of one or more bacterial coding sequences that oxidizehypophosphite to phosphate, and/or phosphite to phosphate, as aselectable marker for the production of transgenic plants.

D. The use of recombinant DNA molecules composed of one or morebacterial coding sequences encoding enzymes that oxidize hypophosphiteto phosphate, and/or phosphite to phosphate, and a promoter sequencefunctional in plants as a selectable marker for the production oftransgenic plants.

E. A chimeric gene functional in a plant cell, which chimeric genecomprises: (1) a plant-expressible promoter sequence; (2) a terminatorsignal sequence; and (3) a coding region of a bacterial gene thatoxidizes phosphite into phosphate, which coding region: encodes afunctional NAD:phosphite oxidoreductase enzyme, and is positionedbetween such plant-expressible promoter sequence and such terminatorsignal sequence so as to be expressible, wherein expression of suchcoding region in a plant cell confers the capacity of using phosphite asa phosphorus source on such plant cell and wherein such capacity to usephosphite as a phosphorus source is capable of providing a basis forselection of such plant cell. The chimeric gene of this paragraph may befurther characterized as follows: (E1)) wherein the coding region isfrom the ptxD gene from Pseudomonas stutzeri, Alcaligenes faecalis, orXanthobacter flavus; (E2) wherein the promoter sequence is aconstitutive promoter; (E3) wherein the promoter sequence is the 35Spromoter from Cauliflower Mosaic Virus; (E4) wherein the terminatorsignal sequence is a nopaline synthetase terminator signal sequence;(E5) wherein the terminator signal sequence is a Cauliflower MosaicVirus terminator signal sequence; or (E6) any combination of E1 throughE5.

F. A transgenic plant that expresses at least one foreign enzyme at alevel enabling the plant to metabolize a reduced form of phosphorus as aphosphorus fertilizer.

Example 9 Selected Embodiments II

This example describes selected embodiments of the invention, presentedas a series of indexed paragraphs.

A. A transgenic plant comprising a construct that confers (1) a growthadvantage on the plant for growth using a reduced form of phosphorus asa nutrient and/or (2) a capability to metabolize at least one reducedform of phosphorus. The transgenic plant of this paragraph may befurther described as follows: (A1) wherein the construct confers agrowth advantage on the plant if phosphite is an at least substantiallyexclusive external source of phosphorus for the plant; (A2) wherein theconstruct confers a growth advantage on the plant if hypophosphite is anat least substantially exclusive external source of phosphorus for theplant; (A3) wherein the construct confers a growth advantage on theplant if phosphite is an at least substantially exclusive externalsource of phosphorus for the plant and if hypophosphite is an at leastsubstantially exclusive external source of phosphorus for the plant;(A4) wherein the transgenic plant is capable of growth without phosphateas an external source of phosphorus, and wherein a non-transgenicvariety of the transgenic plant lacking the construct is at leastsubstantially unable to grow without phosphate as an external source ofphosphorus; (A5) wherein the transgenic plant was transformed initiallywith the construct in a progenitor of the transgenic plant; (A6) whereinthe construct encodes expression of one or more polypeptides that conferon the plant a capability to metabolize at least one reduced form ofphosphorus to phosphate, and, optionally, wherein at least one of thepolypeptides oxidizes phosphite to phosphate, and, optionally, whereinat least one of the polypeptides is capable of using nicotinamideadenine dinucleotide (NAD+) and/or nicotinamide adenine dinucleotidephosphate (NADP+) as an electron acceptor, and, optionally, wherein theone or more polypeptides include a PtxD polypeptide, which, optionally,is encoded by a coding region originating at least substantially fromPseudomonas stutzeri, Alcaligenes faecalis, or Xanthobacter flavus; (A7)wherein the one or more polypeptides of A6 include an HtxA polypeptide;(A8) wherein expression of at least one of the one or more polypeptidesof A6 or A7 is inducible; (A9) wherein expression of at least one of thepolypeptides of any of A6 through A8 is inducible by low phosphate;(A10) wherein expression of at least one of the polypeptides of any ofA6 through A9 is under control of a constitutive promoter; (A11) whereinexpression of at least one of the polypeptides of any of A6 through A10is under control of a leaf-specific promoter; (A12) wherein expressionof at least one of the polypeptides of any of A6 through A11 is undercontrol of a root-specific promoter; (A13) wherein expression of atleast one of the polypeptides of any of A6 through A12 is under controlof a promoter that is not tissue specific; or (A14) any combination ofA1 through A13.

B. A transgenic plant comprising a construct encoding a bacterialpolypeptide that confers on the plant a capability to metabolizephosphite to phosphate.

C. A seed that germinates to produce, or any plant part used to produceor vegetatively reproduce, the transgenic plant of paragraph A or B.

D. A nucleic acid for generating a transgenic plant, comprising: achimeric gene capable of conferring on a plant (1) a growth advantagefor growth using a reduced form of phosphorus as a nutrient and/or (2) acapability to metabolize at least one reduced form of phosphorus. Thenucleic acid of this paragraph may be further described as follows: (D1)wherein the chimeric gene includes a promoter operatively linked to acoding region, and wherein the promoter is capable of controllingexpression of the coding region in a plant, and, optionally, wherein thecoding region encodes one or more polypeptides that oxidize phosphite tophosphate, and, optionally, wherein the coding region is provided atleast substantially by a ptxD gene; (D2) wherein the chimeric geneincludes a promoter operatively linked to a coding region, and whereinthe promoter originated at least substantially in a plant and/or a plantvirus, and, optionally, wherein the promoter includes a 35S promoterfrom Cauliflower Mosaic Virus; (D3) further comprising a transcriptionalterminator that is functional in a plant and operatively linked to thepromoter and the coding region of D1 or D2; (D4) wherein the codingregion of any of D1 through D3 encodes a polypeptide that oxidizesphosphite to phosphate; (D5) wherein the nucleic acid is disposed in amicroorganism; (D6) wherein the nucleic acid is isolated from cells;(D7) wherein the nucleic acid is disposed in a transgenic plant; or (D8)any combination of D1 through D7.

E. A method of generating a transgenic plant, comprising: selecting fortransformation of a plant or plant part using, as a selectable marker, anucleic acid that confers a capability to metabolize a reduced form ofphosphorus. The method of this paragraph may be further described asfollows: (E1) wherein the step of selecting for transformation includesa step of selecting for a growth advantage of the plant or plant part,relative to other plants or plant parts, with one or more reduced formsof phosphorus as an external source of phosphorus for the plants orplant parts; (E2) wherein the step of selecting for a growth advantagein E1 is performed with the plants or plant parts in contact with amedium containing phosphite, hypophosphite, or both; (E3) wherein thestep of selecting for a growth advantage of E1 or E2 is performed withthe medium containing at least substantially no phosphate; (E4), furthercomprising a step of contacting the plant or plant part, a progenitorthereof, or both, with a modifying agent including a construct thatprovides the selectable marker; (E5) wherein the modifying agent of E4includes Agrobacterium cells containing the construct; (E6) wherein theconstruct of E4 or E5 encodes a polypeptide that oxidizes phosphite tophosphate; (E7) wherein the construct of any of E4 through E6 encodes apolypeptide that oxidizes hypophosphite to phosphate; (E8) wherein thestep of contacting of any of E4 through E7 includes a step of firingprojectiles at the plant or plant part, a progenitor thereof, or both;(E9) wherein the step of selecting for transformation is performed witha plant part, and wherein the plant part is a tissue explant or anisolated plant cell; or (E10) any combination of E1 through E9.

F. A method of fertilizing the transgenic plant of paragraph A or B,wherein a reduced form of phosphorus is used as foliar fertilizer oradded to amend soil composition to provide a source of phosphate tosustain plant growth and reproduction.

G. A method of water remediation, comprising: contacting (i) an effluentincluding phosphite and (ii) a transgenic plant comprising a constructthat confers a capability to metabolize phosphite to phosphate, therebyreducing a level of phosphite in the effluent.

Example 10 Selected Embodiments III

This example describes selected embodiments of the invention, presentedas a series of indexed paragraphs.

A. A nucleic acid, comprising: a chimeric gene including (a) a codingregion that encodes a phosphite dehydrogenase enzyme and (b) atranscription promoter operatively linked to the coding region, whereinthe promoter is heterologous with respect to the coding region and isfunctional in plants, fungi, or both, and wherein the chimeric geneprovides sufficient expression of the enzyme, in a plant or fungal cellcontaining the chimeric gene, to confer an ability on the cell tometabolize phosphite (Phi) as a phosphorus source for supporting growth,thereby enabling growth of the cell without an external source ofphosphate (Pi). The nucleic acid of this paragraph may be describedfurther as follows: (A1) wherein the phosphite dehydrogenase enzyme isof bacterial origin; (A2) wherein the phosphite dehydrogenase enzyme isPtxD of Pseudomonas stutzeri (SEQ ID NO:1), an analog or derivative ofPtxD (SEQ ID NO:1), or a PtxD-like homolog from another bacterialspecies; (A3) wherein the bacterial phosphite dehydrogenase enzyme hasan amino acid sequence with at least 50%, 60%, 80%, 90%, or 95% sequenceidentity to at least one of SEQ ID NOS:1-14; (A4) wherein the phosphitedehydrogenase enzyme has an amino acid sequence including a firstsequence region having an NAD-binding motif with sequence similarity oridentity to VGILGMGAIG (SEQ ID NO:15), a second sequence region havingsequence similarity or identity to XPGALLVNPCRGSVVD (SEQ ID NO:16),where X is K or R, a third sequence region having sequence similarity oridentity to GWX₁PX₂X₃YX₄X₅GL (SEQ ID NO.19), where X₁ is R, Q, T, or K,X₂ is A, V, Q, R, K, H, or E, X₃ is L or F, X₄ is G, F, or S, and X₅ isT, R, M, L, A, or S, or includes any combination of the first, second,and third sequence regions; (A5) wherein the phosphite dehydrogenaseenzyme has an amino acid sequence that is at least 90% identical to PtxDfrom Pseudomonas stutzeri (SEQ ID NO:1); (A6) wherein the chimeric genefurther includes a transcription terminator that is operatively linkedto the coding region and heterologous with respect to the coding region;(A7) wherein the promoter is a plant promoter or a viral promoter of aplant virus and is capable of promoting the sufficient expression of theenzyme in a plant cell; (A8) wherein the promoter of A7 corresponds tothe 35S promoter of Cauliflower Mosaic Virus; (A9) wherein the promoterof A7 is inducible by low phosphate availability; (A10) wherein thepromoter of A9 corresponds to a promoter of the PLDZ2 gene ofArabidopsis thaliana; (A11) wherein the chimeric gene is capable ofpromoting the sufficient expression of the enzyme both in a plant celland in a fungal cell each containing the chimeric gene; (A12) whereinthe promoter is a fungal promoter capable of promoting the sufficientexpression of the enzyme in a fungal cell; (A13) wherein one or morecodons of the coding region have been changed in vitro to improvetranslational efficiency in plants and/or fungi; (A14) furthercomprising an intron connected to the coding region and configured to betranscribed with the coding region and removed by splicing aftertranscription, wherein the intron is optionally disposed within thecoding region; (A15) wherein the coding region has at least 90% sequenceidentity with SEQ ID NO:21; or (A16) any combination of A1 through A15.

B. A plant cell comprising a nucleic acid that expresses a phosphitedehydrogenase enzyme in the plant cell and capable of metabolizingphosphite as a source of phosphorus for supporting growth. Optionally,the nucleic acid is according to paragraph A. The plant cell of thisparagraph may be described further as follows: (B1) further comprisingan other nucleic acid that expresses a hypophosphite dehydrogenaseenzyme, optionally of bacterial origin, in the plant cell; (B2) theplant cell of B1 wherein the nucleic acids collectively confer anability on the cell to metabolize hypophosphite (Hphi) as a phosphorussource for supporting growth; (B3) wherein the other nucleic acid of B1or B2 encodes a polypeptide with at least 95% sequence identify to HtxAof Pseudomonas stutzeri (SEQ ID NO:20); (B4) the plant cell of any of B1through B3, wherein the nucleic acids are integrated adjacent oneanother in the genome of the plant cell; (B5) wherein expression of thephosphite dehydrogenase enzyme, the hypophosphite dehydrogenase enzyme,or both are controlled by a root-specific promoter; (B6) wherein theplant cell is homozygous for the nucleic acid; (B7) wherein the plantcell is a eukaryotic algal cell; (B8) wherein the algal cell of B7 is aChlamydomonas cell; (B9) wherein the plant cell is from a species ofvascular plant; or (B10) any combination of B1 through B9.

C. A plant composed of a plurality of plant cells according to paragraphB. The plant of this paragraph may be described further as follows: (C1)wherein the plant is a vascular plant, such as a species of crop plant,(C2) wherein the species of crop plant of C1 is selected from the groupconsisting of maize, soybean, rice, potatoes, tomatoes, sugarcane, andwheat.

D. A fungal cell comprising a nucleic acid that expresses a phosphitedehydrogenase enzyme in the fungal cell and capable of metabolizingphosphite as a source of phosphorus for supporting growth. Optionally,the nucleic acid is according to paragraph A. The fungal cell of thisparagraph may be described further as follows: (D1) further comprising anucleic acid that expresses a bacterial hypophosphite dehydrogenaseenzyme in the fungal cell; (D2) the fungal cell of D1, wherein thenucleic acids collectively confer an ability on the cell to metabolizehypophosphite (Hphi) as a phosphorus source for supporting growth of thefungal cell; (D3) wherein the fungal cell is from a species ofTrichoderma; (D4) wherein the fungal cell is a member of a species ofmycorrhizal fungus capable of forming a symbiotic relationship with aplant; or (D5) any combination of D1 through D4.

E. A method of reducing fungal infections in plants, comprising:applying a plurality of the fungal cells of paragraph D to a seed formof plants, the plants themselves, soil in which the plants are disposed,or a combination thereof. In some cases, the fungal cells may be spores.

F. A plant associated with a plurality of fungal cells according toparagraph D to form mycorrhizae. Optionally, the fungal cells render theplant capable of growing on a medium containing phosphite (Phi),hypophosphite (Hphi), or both, as a phosphorus source for supportinggrowth.

G. A method of fertilizing a crop plant using hypophosphite and/orphosphite as a phosphorus source for supporting growth, the crop plant(a) including a plurality of cells comprising the nucleic acid ofparagraph A, (b) forming mycorrhizae with a mycorrhizal funguscomprising the nucleic acid of paragraph A, and/or (c) being associatedwith a Trichoderma fungus comprising the nucleic of claim 1, the methodcomprising: applying at least one reduced form of phosphorus to theplant and/or to soil adjacent the plant, such that the reduced form ismetabolized to phosphate by the plant and/or the fungus to supportgrowth and productivity of the plant.

H. A method of fertilizing the plant of paragraph C, the methodcomprising: applying at least one reduced form of phosphorus to theplant and/or to soil adjacent the plant, such that the reduced form ismetabolized to phosphate by the plant to support growth and productivityof the plant. Optionally, the reduced form may be applied as foliarfertilizer or added to amend soil to provide a source of phosphate tosustain plant growth and reproduction of the plant.

I. A method of treating water to lower its content of reducedphosphorus, the method comprising: contacting water containinghypophosphite and/or phosphite with a plurality of the plant cellsand/or fungal cells comprising the nucleic acid of paragraph A, suchthat at least a portion of the hypophosphite and/or phosphite isoxidized to phosphite and/or phosphate. Optionally, the step ofcontacting includes a step of contacting the water with a plurality ofvascular plants composed of plant cells comprising the nucleic acid ofparagraph A.

J. A method of treating liquid waste to lower its content of reducedphosphorus, the method comprising: contacting (i) water containinghypophosphite and/or phosphite as a contaminant and (ii) a plurality ofthe plant cells and/or fungal cells comprising the nucleic of paragraphA, such that at least a portion of the hypophosphite and/or phosphite isoxidized to phosphite and/or phosphate.

K. A method of utilizing the nucleic acid of paragraph A for productionof a transgenic plant, comprising: selecting for growth of plant cellscomprising the nucleic acid of paragraph A as a selectable marker duringproduction of a transgenic plant.

L. A method of obtaining a plant transformed with a nucleic acidencoding a phosphite dehydrogenase enzyme that is expressible from thenucleic acid as a selectable marker, comprising: contacting plant cellsand a composition including the nucleic acid under conditions thatpromote introduction of the nucleic acid into at least a subset of theplant cells; culturing the plant cells in a medium containing phosphiteas a primary or exclusive phosphorus source for growth; selectingtransformed plant cells produced by the steps of contacting andculturing, and expressing the phosphite dehydrogenase enzyme asevidenced by growth in the medium; and regenerating at least a portionof the transformed plant cells into a transgenic plant. The method ofthis paragraph may be described further as follows: (L1) wherein thecomposition includes Agrobacterium cells that supply the nucleic acidduring the step of contacting; or (L2) wherein the composition includesprojectiles that are fired at the plant cells in the step of contacting.

M. A plant, comprising: a nucleic acid including a chimeric geneexpressing a phosphite dehydrogenase enzyme such that the plant iscapable of metabolizing phosphite (Phi) as a phosphorus source forsupporting growth, thereby enabling growth of the plant without anexternal source of phosphate (Pi). The plant of this paragraph may bedescribed further as follows: (M1) wherein the nucleic acid is stablyintegrated into the genome of the plant; (M2) wherein the plant is avascular plant; (M3) wherein the plant is a species of algae; (M4)wherein the phosphite dehydrogenase enzyme has any of the features ofparagraph A; or (M5) any combination of M1 through M4.

N. A fungus, comprising: a nucleic acid including a chimeric geneexpressing a phosphite dehydrogenase enzyme such that the fungus iscapable of metabolizing phosphite (Phi) as a phosphorus source forsupporting growth, thereby enabling growth of the fungus without anexternal source of phosphate (Pi). The fungus of this paragraph may bedescribed further as follows: (N1) wherein the nucleic acid is stablyintegrated into the genome of the fungus; (N2) wherein the fungus is aspecies of Trichoderma; (N3) wherein the fungus is a mycorrhizal speciescapable of forming a symbiotic relationship with a plant; (N4) furthercomprising a plant associated with the fungus to form mycorrhizae; (N5)wherein the phosphite dehydrogenase enzyme has any of the features ofparagraph A; or (N6) any combination of N1 through N5.

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in applications claiming priority from this or a relatedapplication. Such claims, whether directed to a different invention orto the same invention, and whether broader, narrower, equal, ordifferent in scope to the original claims, also are regarded as includedwithin the subject matter of the inventions of the present disclosure.

1. A biological cell, comprising: a fungal cell transgenically adaptedto express an enzyme that catalyzes oxidation of phosphite to phosphate.2. The biological cell of claim 1, wherein the enzyme is a phosphitedehydrogenase.
 3. The biological cell of claim 1, wherein the phosphitedehydrogenase is of bacterial origin.
 4. The biological cell of claim 2,wherein the phosphite dehydrogenase is PtxD of Pseudomonas stutzeri (SEQID NO:1), an analog or derivative of PtxD of SEQ ID NO:1, or a PtxD-likehomolog.
 5. The biological cell of claim 4, wherein the phosphitedehydrogenase has an amino acid sequence with at least 80% sequenceidentity to at least one of SEQ ID NOS:1-14.
 6. The biological cell ofclaim 1, wherein the enzyme has an amino acid sequence including a firstsequence region having an NAD-binding motif with sequence similarity oridentity to VGILGMGAIG (SEQ ID NO:15), a second sequence region havingsequence similarity or identity to XPGALLVNPCRGSVVD (SEQ ID NO:16),where X is K or R, and a third sequence region having sequencesimilarity or identity to GWX₁PX₂X₃YX₄X₅GL (SEQ ID NO.19), where X₁ isR, Q, T, or K, X₂ is A, V, Q, R, K, H, or E, X₃ is L or F, X₄ is G, F,or S, and X₅ is T, R, M, L, A, or S.
 7. The biological cell of claim 1,wherein the enzyme has an amino acid sequence that is at least 90%identical to PtxD from Pseudomonas stutzeri (SEQ ID NO:1).
 8. Thebiological cell of claim 1, wherein the fungal cell is a species ofTrichoderma.
 9. The biological cell of claim 1, wherein the fungal cellis a mycorrhizal species capable of forming a symbiotic relationshipwith a plant.
 10. The biological cell of claim 1, wherein the fungalcell is transformed with a nucleic acid construct including a fungalpromoter operatively linked to a coding sequence for the enzyme.
 11. Thebiological cell of claim 10, wherein the nucleic acid construct includesa transcription terminator operatively linked to the fungal promoter andthe coding sequence.
 12. A nucleic acid, comprising: a chimeric geneincluding (a) a coding region that encodes an enzyme that catalyzesoxidation of phosphite to phosphate and (b) a transcription promoteroperatively linked to the coding region, wherein the chimeric geneprovides sufficient expression of the enzyme, in a fungal cellcontaining the chimeric gene, to confer an ability on the fungal cell tometabolize phosphite as a phosphorus source for supporting growth. 13.The nucleic acid of claim 12, wherein the enzyme is a phosphitedehydrogenase.
 14. The nucleic acid of claim 13, wherein the phosphitedehydrogenase is of bacterial origin.
 15. The nucleic acid of claim 13,wherein the phosphite dehydrogenase is PtxD of Pseudomonas stutzeri (SEQID NO:1), an analog or derivative of PtxD of SEQ ID NO:1, or a PtxD-likehomolog.
 16. The nucleic acid of claim 15, wherein the enzyme has anamino acid sequence with at least 80% sequence identity to at least oneof SEQ ID NOS:1-14.
 17. The nucleic acid of claim 12, wherein thetranscription promoter is from a fungus.
 18. A method of propagating afungus, comprising: obtaining a fungus transgenically adapted to expressan enzyme that catalyzes oxidation of phosphite to phosphate; andgrowing the fungus in a medium containing phosphite as a source ofphosphorus.
 19. The method of claim 18, wherein the enzyme is aphosphite dehydrogenase.
 20. The method of claim 18, wherein phosphiteis a predominant or exclusive source of phosphorus in the medium.
 21. Amethod of plant fertilization, comprising: obtaining a fungus that hasbeen transgenically modified to express an enzyme that catalyzesoxidation of phosphite to phosphate; associating the fungus and theplant with each other; and growing the plant associated with the fungusin a presence of phosphite such that the fungus converts the phosphiteto phosphate to support growth of the plant.
 22. The method of claim 21,wherein the step of associating includes a step of a step of creatingcontact between the transgenically modified fungus and a seed of theplant.
 23. The method of claim 21, wherein the step of associatingincludes a step of disposing the transgenically modified fungus in soiladjacent the plant.
 24. The method of claim 21, wherein the step ofassociating includes a step of applying the transgenically modifiedfungus to the plant.
 25. The method of claim 21, wherein the fungus is aspecies of Trichoderma.